Fluorescent fusion polypeptides and methods of use

ABSTRACT

Embodiments of the present invention provide for the facile generation of a stable recombinant fusion polypeptides with intrinsic fluorescent properties. The recombinant antibodies may be suitable for qualitative and/or quantitative immunofluorescence analysis. Generally, the fluorescent polypeptides include a fluorescent domain comprising a C-terminus and an N-terminus; a first antibody domain covalently linked to the C-terminus; and a second antibody domain covalently linked to the N-terminus.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 13/096,695, filed Apr. 28, 2011, now U.S. Pat. No. 8,877,898,which claims priority to U.S. Provisional Patent Application Ser. No.61/343,627, filed Apr. 30, 2010, each of which is hereby incorporated byreference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under R01 AI066045awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Nanotechnology and synthetic biology are converging areas with thepotential to create novel drugs and diagnostic molecules, and to inspirea plethora of innovative applications exemplified by the advances madein the area of antibody engineering since the late 1980s. Recentlyefforts have been directed towards creating antibody fragments withnovel effector functions as fusions with fluorescent proteins. Thesehave used scFv fused directly to the fluorescent protein either at theC-terminus or the N-terminus, resulting in additive properties of thescFv and the fluorophore. Alternative efforts have produced a greenfluorescent protein (GFP) scaffold that retains fluorophore activity andis capable of accommodating two proximal, randomized binding loops.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a polypeptide that generallyincludes a fluorescent domain that possesses a C-terminus and anN-terminus, a first antibody domain covalently linked to the C-terminus,and a second antibody domain covalently linked to the N-terminus.

In some embodiments, at least one covalent link comprises a linkercomprising no more than 10 amino acids.

In some embodiments, at least one of the fluorescent domain, the firstantibody domain, or the second antibody comprises an affinity tag.

In some embodiments, the first antibody domain and the second antibodydomain specifically bind to a single target molecule, while in otherembodiments, the first antibody domain and the second antibody domainspecifically bind to different target molecules.

In some embodiments, the first antibody domain comprises a variablelight chain (V_(L)) comprising an N-terminus. In some embodiments, thesecond antibody domain comprises a variable heavy chain (V_(H))comprising a C-terminus. In some embodiments, the polypeptide caninclude both a first antibody domain comprises a variable light chain(V_(L)) comprising an N-terminus and a second antibody domain comprisesa variable heavy chain (V_(H)) comprising a C-terminus. In certain ofthese embodiments, the N-terminus of first antibody domain andC-terminus of the second antibody domain are separated by a distance ofno less than 30 Å and no more than 40 Å.

In some embodiments, the fluorescent domain comprises at least a portionof a monomeric fluorescent protein sufficient to emit a fluorescentsignal.

In some embodiments, the fluorescent domain comprises at least a portionof a dimeric fluorescent protein sufficient to emit a fluorescentsignal.

In some embodiments, the fluorescent domain comprises an amino acidsequence comprising at least 90% amino acid sequence similarity to atleast one of: amino acids 122-338 of SEQ ID NO:2, amino acids 3-219 ofSEQ ID NO:4, amino acids 3-235 of SEQ ID NO:6, or amino acids 3-235 ofSEQ ID NO:8.

In some embodiments, the fluorescent domain comprises an amino acidsequence comprising at least 90% amino acid sequence identity to atleast one of: amino acids 122-338 of SEQ ID NO:2, amino acids 3-219 ofSEQ ID NO:4, amino acids 3-235 of SEQ ID NO:6, or amino acids 3-235 ofSEQ ID NO:8.

Moreover, in certain embodiments, the polypeptide can include featuresof any combination of two or more embodiments summarized above.

In another aspect, the invention provides a composition that generallyincludes two or more different fluorescent fusion polypeptidessummarized above.

In some embodiments, such a composition can include a first fluorescentfusion protein that includes a first fluorescent domain and a secondfluorescent fusion protein that includes a second fluorescent domain. Incertain of these embodiments, the first fluorescent domain can emit afirst fluorescent signal and the second fluorescent domain can emit asecond fluorescent signal. In some of these embodiments, the firstfluorescent signal comprises a first emission peak, the secondfluorescent signal comprises a second emission peak, and the firstemission peak is different than the second emission peak.

In some embodiments, a first fluorescent polypeptide specifically bindsto a different target molecule than a second fluorescent fusionpolypeptide.

In another aspect, the invention provides a polynucleotide that encodesany one of the polypeptides summarized above.

In another aspect, the invention provides a cell that includes such apolynucleotide.

In some embodiments, the cell can further include a secondpolynucleotide that encodes a second polypeptide as summarized above. Insome of these embodiments, the fluorescent domain of one polypeptide canemit a first fluorescent signal comprising a first emission peak, thefluorescent domain of the second polypeptide can emit a secondfluorescent signal comprising a second emission peak, and the firstemission peak can be different than the second emission peak.

In another aspect, the invention provides a cell that includes any oneof the polypeptides summarized above. In some of these embodiments, thecell may be a microbe, while in other embodiments, the cell may be ananimal cell such as, for example, a tumor cell.

In another aspect, the invention provides a cell that includes the anyone of the compositions summarized above. In some of these embodiments,the cell may be a microbe, while in other embodiments, the cell may bean animal cell such as, for example, a tumor cell.

In another aspect, the invention provides a method that generallyincludes introducing into a cell any one of the polynucleotidessummarized above. In some of these embodiments, the cell may be amicrobe, while in other embodiments, the cell may be an animal cell suchas, for example, a tumor cell.

In another aspect, the invention provides a method that generallyincludes releasing a microbe into an environment, wherein the microbecomprises any one of the polynucleotides summarized above and at leastone additional heterologous polynucleotide, and detecting the microbe bydetecting the fluorescent signal. In some embodiments, the method caninclude detecting the fluorescent signal at a plurality of time points.

In another aspect, the invention provides a method that generallyincludes releasing a microbe into an environment inhabited by apathogen, wherein the microbe include a polynucleotide summarized abovethat encodes a polypeptide that interferes with transmission of thepathogen, and permitting the cell to express and export the polypeptideencoded by the polynucleotide summarized above so that the polypeptideinterferes with transmission of the pathogen. In some of theseembodiments, the exported polypeptides binds to the pathogen. In otherof these embodiments, the exported polypeptide binds to a compoundproduced by the pathogen. In some embodiments, the microbe is a symbiontof a host organism, which is further a host organism to the pathogen. Incertain embodiments, the method can further include detecting afluorescent signal produced by the fluorescent domain of the polypeptideencoded by the polynucleotide summarized above. In some embodiments, themethod can include detecting the fluorescent signal at a plurality oftime points.

In another aspect, the invention provides a method that generallyincludes detecting a fluorescent signal produced by cell, wherein thecell comprises a polynucleotide as summarized above that encodes apolypeptide comprising a fluorescent domain. In some embodiments, themethod further includes detecting the fluorescent signal before, during,or after surgery to remove tumor cells emitting the fluorescent signal.In some embodiments, the method includes detecting the fluorescentsignal at a plurality of time points. In some embodiments, the methodincludes exposing the cell to white light under conditions effective forthe fluorescent signal to generate an amount of a reactive oxygenspecies effective to kill the cell.

In another aspect the invention provides a method that generallyincludes administering to a subject any one of the polypeptidessummarized above, wherein at least one antibody domain of thepolypeptide specifically binds to a marker of inflammation, anddetecting a fluorescent signal produced by the fluorescent domain of thepolypeptide. In some embodiments, the method includes detecting thefluorescent signal at a plurality of time points. In some embodiments,detecting the fluorescent signal may be performed non-invasively.

In another aspect, the invention provides a method that generallyincludes providing a sample that comprises the analyte, contacting thesample with any one or more of the polypeptides summarized above,wherein at least one antibody domain specifically binds to the analyte,removing unbound polypeptides, and detecting a fluorescent signalproduced by the polypeptide specifically bound to the analyte, therebydetecting presence of the analyte in the sample. In some embodiments,the method can further include immobilizing at least a portion of thesample on a substrate. In some embodiments, the method can furtherinclude contacting at least a portion of the sample with a secondpolypeptide as summarized above, wherein at least one antibody domain ofthe second polypeptide specifically binds to the second analyte removingunbound second polypeptide, and detecting fluorescent signal produced bythe second polypeptide specifically bound to the second analyte, therebydetecting presence of the second analyte in the sample. In someembodiments, the method can further include quantifying the fluorescentsignal.

In another aspect, the invention provides a kit that generally includesa first container comprising a first polypeptide as summarized abovecomprising a first fluorescent domain that produces a fluorescent signalcomprising a first emission peak, and at least one antibody domain thatspecifically binds to a first analyte and a second container comprisinga second polypeptide as summarized above comprising a second fluorescentdomain that produces a fluorescent signal comprising a second emissionpeak, and at least one antibody domain that specifically binds to asecond analyte.

In another aspect, the invention provides a method of making a fusionpolypeptide. Generally, the method includes creating an expressionvector that comprises a polynucleotide operably linked to a promoter,wherein the polynucleotide encodes an fusion polypeptide comprising: afluorescent domain comprising a C-terminus and an N-terminus, a firstantibody domain covalently linked to the C-terminus, and a secondantibody domain covalently linked to the N-terminus; introducing theexpression vector into a host cell; and growing the host cell comprisingthe expression vector in conditions effective for the host cell toexpress the fusion polypeptide.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Ribbon representation of a REDantibody molecule 3D model. TheV_(H) chain is shown in green, RFP domain in red, the V_(L) chain inyellow. His-tag is shown in blue and linkers between V_(H)-RFP andRFP-V_(L) are in cyan with the distances indicated. Antigen binding siteis indicated as a black circle on antibody structure. All distances areshown as a grey lines and are indicated in the angstroms (A).

FIG. 2: Generation of expression plasmid (A) and E. coli expressioncassette (B) of REDantibody. (A) The single chain Fv encoding fragmentflanked by NcoI and NotI sites with an inframe BamHI site between theV_(H) and V_(L) was inserted into pBAK1 vector to generate pBAK1B72.3,pBAK1CA19.9 and pBAK14D5. The mRFP encoding fragment flanked by BamHIsites was inserted into the pBAK1B72.3, CA19.9 and 4D5 to generatepBAK172.3RFP, pBAK1CA19.9RFP and pBAK14D5RFP. (B) The expressioncassette has a T7 promoter to initiate transcription using T7 RNApolymerase, lac operator is for the control of transcription using IPTG.RBS is ribosome binding site where translation begins, pelB sequence toguide newly synthesized molecule into the E. coli periplasm where thissequence is cleaved and disulphide bonds are formed. V_(H) is variableheavy chain of antibody, Link is an additional 5 amino acids to flankeither side of the mRFP1 gene, mRFP is a gene of red fluorescent proteinand V_(L) is variable light domain of antibody. This is followed byHis-tag sequence for immobilized metal affinity purification and stopcodon. T7 terminator is to terminate transcription by T7 RNA polymerase.

FIG. 3: Expression and purification (A) CA19.9 scFv and (B) CA19.9REDantibody. The purified savCA19.9 (A) and REDantibodyCA19.9 (B) wereresolved by 12% SUS-PAGE and stained with Coomassie Brilliant Blue R250.Lane 1: molecular weight markers, 70, 40, 30 and 20 kDa; lane 2: Ni-NTAcolumn elution 1; lane 3: Ni-NTA column elution 2; lane 4: Ni-NTA columnelution 3; lane 5: Ni-NTA column elution 4.

FIG. 4: Gel filtration on Sephadex G200 column. Panel A—standardproteins (peak1—200 kDa, peak 2—66 kDa, peak 3—29 kDa, peak 4—12.4 kDa);Panel B—mRFP1 and panel C REDantibodyCA19.9.

FIG. 5: REDantibody expression and purification. (A) E. coli cellsexpressing REDantibody (A1) and scFv (A2) resuspended in periplasmicbuffer, (B) periplasmic fraction of extracted REDantibody (B1) and scFvantibody (B2) and (C) is REDantibody fractions 1-5 eluted from theNi-NTA column and labeled as C1-05 respectively.

FIG. 6: Immunofluorescent microscopic analysis of T. cruzi epimastigotesstained with REDantibody CA19.9 (A), REDantibody B72.3 (B) and negativecontrol REDantibody 4D5 (C). T. cruzi epimastigote is stained withREDantibody CA19.9 and viewed by microscopy reveals a maximum intensityfluorescent image (D). The surface staining combined with confocalmicroscopy reveals a slight invagination on the parasite surface (E). Acut-away of the image (E) reveals the staining is limited to the surfaceof the T. cruzi epimastigote (F).

FIG. 7 shows the PCR primers used in certain exemplary embodiment.

FIG. 8A-B shows plasmid maps and 3D model of genetically encodedfluorescent antibody. (A) Molecular model (ribbon representation) of theHERCEPTIN V_(H) and V_(L) (Genentech, Inc., South San Francisco, Calif.)antigen-binding fragment (Fv) complexed with human HER2 (PDB 1N8Z) withmRFP1 linker based on the DsRed (PDB 1G7K). A portion of HER2 is shownin orange, V_(H) chain of 4D5-8 in green, mRFP1 in red and V_(L) chainof 4D5-8 antibody in navy blue. (B) Plasmid maps of pBAK1 vector (FIG.8B-1), p4D5-8Bam (FIG. 8B-1) and p4D5-8mRFP1 (FIG. 8B-2) used to cloneand express red fluorescent antibody. The expression cassette has a T7promoter region, a lac operator, a ribosome-binding site followed by acloning region with NdeI, NcoI and NotI restriction sites for theinsertion of the DNA sequences. At the end, there is a T7 terminationsequence to restrict translation to the expression of RNA for therecombinant protein. The vector also has the kanamycin resistance gene,ColE1 pBR322 origin of replication and lad repressor gene. The4D5-8mRFP1 construct starts with pelB leader sequence to direct theprotein to the periplasmic space of E. coli where it is cleaved off. Themature protein begins with V_(H) chain, followed by a 5-amino acidlinker, mRFP1 sequence, second 5-amino acid linker, V_(L) chain andterminates with an octa-His-tag.

FIG. 9 shows purification and characterization of the 4D5-8mRFP1recombinant protein. (a) SDS-PAGE and Coomassie stained gel of4D5-8mRFP1: molecular markers (Lane 1), uninduced sample (Lane 2), IPTGinduced sample (Lane 3), soluble fraction (Lane 4), flow-throughNi²⁺-nitrilotriacetic acid column (Lane 5), wash fraction (Lane 6),elution fractions using 500 mM concentration imidazole (Lanes 7-10). (b)Western blotting of SDS-PAGE gel of 4D5-8mRFP1 (as in panel a) analyzedusing HRP-labelled anti-His-tag antibody. (c) Gel filtrationchromatography of standards proteins (peaks 1-4) and Ni²⁺nitrilotriacetic acid purified 4D5-8mRFP1 protein using Sephadex G 200gel bead column. (d) SDS-PAGE and Coomassie stained gel of gelfiltration chromatography 4D5-8mRFP1 (pooled fractions 32-37): molecularmarkers (Lane 1), mRFP1 (Lane 2) 4D5-8mRFP1 (Lane 3).

FIG. 10 shows binding properties of purified 4D5-8mRFP1. (a) Detectionof 4D5-8mRFP1 binding to p185^(HER-2-ECD)-overexpressing SKBr-3 cells byflow cytometry. Cells incubated with 4D5-8mRFP1 are shown as filled peakand cells used as controls (solid line) were treated with MACS bufferalone. All experiments were normalized to 20,000 cells, excludingdoublets. (b) Flow cytometry analysis of p185^(HER-2-ECD) non expressingMDA-MD-231 cells incubated with 4D5-8mRFP1. (c) Fluorescence images ofSKBr-3 cells incubated with 4D5-8mRFP1 and DNA (nucleus) binding dyeGelGreen™ and (d) Bright-field images of the same SKBr-3 cells incubatedwith 4D5-8mRFP1.

FIG. 11 shows the nucleic acid (SEQ ID NO:1) and amino acid sequence(SEQ ID NO:2) for the V_(H), V_(L), and fluorophore of B72.3RFP. Thetertiary structure is illustrated in FIG. 1. Gray shaded amino acidscorrespond to the V_(H) domain; bold text amino acids correspond to thefluorescent domain (mRFP domain); and underlined amino acids correspondto the V_(L) domain.

FIG. 12 shows the nucleic acid (SEQ ID NO:3) and amino acid sequence(SEQ ID NO:4) of mRFP1. The fluorescent domain is reflected in aminoacids 3-219 of SEQ ID NO:4.

FIG. 13 shows the nucleic acid (SEQ ID NO:5) and amino acid sequence(SEQ ID NO:6) of mCitrine. The fluorescent domain is reflected in aminoacids 3-235 of SEQ ID NO:6.

FIG. 14 shows the nucleic acid (SEQ ID NO:7) and amino acid sequence(SEQ ID NO:8) of mCerulean. The fluorescent domain is reflected in aminoacids 3-235 of SEQ ID NO:8.

FIG. 15: (A) Excitation and emission peaks of REDantibody 4D5-8. (B)Detection of REDantibody 4D5-8 based on concentration dependent emissionat 607 nm.

FIG. 16A-E shows plasmid maps of vectors used to express 4D5-8antibodies. Fluorescent proteins were inserted in-between theV_(H)/V_(L) regions of 4D5-8. A) The plasmid map for parent plasmidpA4D5-81. B) The plasmid map and expression cassette for constructpA4D5-8mRFP. C) The plasmid map for construct pA4D5-81-mCit. D) Theplasmid map for construct pA4D5-81-mCer. E) The plasmid map forconstruct pA4D5-81-BFP.

FIG. 17A-D shows excitation and emission spectra of 4D5-8 fluorescentfusion antibodies 4D5-8_mRFP (A), 4D5-8_mCit (B), 4D5-8_Cerul (C), and4D5-8_BFP (D).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The present invention involves novel fusion proteins constructs,polynucleotides that encode the constructs, and methods, includingtherapeutic, diagnostic, and detection methods, employing suchconstructs.

We describe herein fusion proteins that include a fluorescent proteindomain linked to one or more antibody domains. In one exemplaryembodiment, illustrated in FIG. 1, we used monomeric red fluorescentprotein (mRFP) derived from Discosoma to link the V_(H) and V_(L) domainpairing of the recombinant anti-carbohydrate antibodies B72.3, CA19.9,and 4D5 anti-p185HER2.

Many antibody structures have been determined by X-ray crystallography.These analyses have revealed that the native distance between theC-terminus on the variable heavy chain and the N-terminus on thevariable light chain is often approximately 35 Å. To generateconventional single chain fragment of the variable region (scFv)antibodies, a 20-30 amino acid linker may be introduced between thesetwo sites and thus provide a flexible region of approximately 35 Å. Thisspacing between the V_(H) and V_(L) can influence the functionality ofthe scFv because the non-covalent interactions between V_(H)/V_(L)interfaces are involved in antigen recognition. Nevertheless, theV_(H)/V_(L) pairing exist in equilibrium with the unpaired state, oftenresulting in aggregation of the variable chains and, therefore, reducedantigen recognition and decreased stability relative to the Fab fragmentor whole immunoglobulin.

We have engineered a fusion polypeptide that uses a β-barrel fluorescentdomain to bridge the V_(H) and the V_(L) and enhance stability of theantibody domains by maintaining the correct spatial geometry between theantibody domains. Moreover, the fluorescent domain anchors theN-terminus and the C-terminus on the same plane with similar spatialdimension to the Fv in the context of a Fab fragment so that appropriateV_(H)/V_(L) interface interactions may be achieved resulting infunctional binding sites.

Generally, prior antibody-fluorophore conjugates have been establishedas valuable tools in basic and applied research, despite havinglimitations of photobleaching, batch variation, Fc receptor binding,aggregation, and partial loss of binding. Some of these issues can beaddressed by using recombinant single-chain fragment variable antibodyfused directly to genetically encoded fluorescent reporters. However,conventional single-chain fragment variable domains linked by longflexible linkers are themselves prone to disassociation and aggregation.

Here we report the design, assembly, bacterial production, andpurification of a modular model that may be used generally to producenovel antibody-fluorescent protein fusion molecules. In one particularembodiment, the insertion of fluorescent protein mRFP1 in-between theV_(H)/V_(L) regions of anti-p185HER2-ECD antibody 4D5-8 resulted inoptimal V_(H)/V_(L) interface interactions to create 4D5-8 REDantibody.The bacterially expressed monomeric molecule used in flow cytometry andcell staining studies with SKBr-3 cells retained the fluorophoreproperties and antibody specificity functions. The molecular model maybe generalized beyond the use of mRFP1 and the 4D5-REDantibody and may,instead, use the fluorescent domain of any suitable fluorescent protein,as described in more detail below. Thus, references to REDantibody aremerely an exemplary and should not be construed as limiting.

Thus, in one aspect, the invention provides a fusion polypeptide thatincludes a fluorescent domain comprising a C-terminus and an N-terminus,a first antibody domain covalently linked to the C-terminus, and asecond antibody domain covalently linked to the N-terminus.

In some embodiments, the polypeptide can include a linker that providesthe covalent linkage between the fluorescent domain and one of theantibody domains. In such embodiments, the polypeptide can include twolinkers so that a linker provides the link between the fluorescentdomain and each of the antibody domains. The linker may be of anysuitable length. In some embodiments, the linker assists in providingthe stability of the molecule by contributing to spacing between theC-terminus of one antibody domain and the N-terminus of the secondantibody domain that reflects the spacing natively found in a Fabfragment or full IgG. In such embodiments, the length of the linker maybe no more than 10 amino acids such as, for example, no more than nineamino acids, no more than eight amino acids, no more than seven aminoacids, no more than six amino acids, no more than five amino acids, nomore than four amino acids, no more then three amino acids, or no morethan two amino acids. When two linkers are present, the length of onelinker may be identical or different than the length of the otherlinker. In one embodiment, the linker can include five amino acids suchas, for example, Gly-Gly-Gly-Gly-Ser_(SEQ ID NO:21).

As used herein, an antibody domain is that portion of the fusionpolypeptide that exhibits affinity interaction with a ligand that is tosome degree specific. As used herein, “specific” and variations thereofrefer to having a differential or a non-general affinity, to any degree,for a particular target. In some embodiments, an antibody domain caninclude any suitable portion of an immunoglobulin. As immunoglobulinstructure and function are well characterized, those of skill in the artare well equipped to determine the amount and the particular portions ofan immunoglobulin necessary to provide desired target recognition. Insome embodiments, an antibody domain can include, for example, a V_(L)or a V_(H) chain. In some of these embodiments, one antibody domain caninclude a V_(L) chain while the second antibody domain can include aV_(H) chain. In particular embodiments, a V_(L) chain can be covalentlylinked to the C-terminus of the fluorescent domain and a V_(H) chain canbe covalently linked to the N-terminus of the fluorescent domain.

In some embodiments, the terminus of the antibody domains that arecovalently linked to the fluorescent domain—either directly or via alinker, if present—may be separated by a predetermined distance. Theminimum predetermined distance may be at least 30 Å such as, forexample, at least 31 Å, at least 32 Å, at least 33 Å, at least 34 Å, atleast 35 Å, at least 36 Å, at least 37 Å, at least 38 Å, or at least 39Å. The maximum predetermined distance may be no more than 40 Å such as,for example, no more than 39 A, no more than 38 Å, no more than 37 Å, nomore than 36 Å, no more than 35 Å, no more than 34 Å, no more than 33 Å,no more than 32 Å, or no more than 31 Å. The predetermined distance maybe within a range defined by any minimum and any maximum distancedescribed herein. Also, in certain embodiments, the predetermineddistance may be any of the minimum or maximum endpoints describedherein. Thus, the predetermined distance may be 30 Å, 31 Å, 32 Å, 33 Å,34 Å, 35 Å, 36 Å, 37 Å, 38 Å, 39 Å, or 40 Å.

In some embodiments, the antibody domains may specifically bind to thesame or to different target molecules. Thus, embodiments in which bothantibody domains specifically bind to the same target molecule can becharacterized as mono-specific. In contrast, a fluorescent fusionpolypeptide that includes antibody domains that specifically bind todifferent target molecules may be characterized as bi-specificpolypeptides.

The antibody domains may be, or be derived from, any suitable antibody.For example, we report herein producing a fluorescent fusion polypeptidethat includes antibody domains derived from antibodies that specificallybind to human breast cancer markers (FIG. 8 through FIG. 10) as well asa fluorescent fusion polypeptide that includes antibody domains derivedfrom antibodies that specifically bind to T. cruzi markers (FIG. 2through FIG. 6). Other exemplary antibody domains can include, forexample, CA125 anti-MUC16, H23 anti-MUC1 and 1C3 anti-Plasmodiumfalciparum chitinase. The modular nature of the fluorescent fusionpolypeptides described herein permit great design flexibility and thepossibility of exploiting the known binding affinity and specificity ofany known antibody.

The fluorescent domain includes at least a portion of any suitablefluorescent polypeptide sufficient to emit a fluorescent signal. Thestructure and function of fluorescent polypeptides are wellcharacterized. Thus, a person of ordinary skill in the art can readilydetermine the portion of a fluorescent polypeptide that is required tomaintain fluorescent functionality.

In some embodiments, the fluorescent domain can optionally providestructural integrity to the molecule in addition to providing a sourcefor the fluorescent signal. Thus, in some embodiments, the fluorescentdomain can include a portion of the fluorescent polypeptide sufficientto emit a fluorescent signal and to provide desired steric stability.

In some embodiments, the fluorescent fusion polypeptide can include atleast a portion of a monomeric fluorescent protein sufficient to emit afluorescent signal. Many monomeric fluorescent polypeptides possess aβ-barrel structure that is capable of maintaining a desiredpredetermined distance between the C-terminus of one antibody domain andthe N-terminus of the second antibody domain.

Portions of certain dimeric fluorescent polypeptides also may suitablefor use as the fluorescent domain. Dimeric fluorescent polypeptides maynaturally orient so that they can provide steric stability as describedabove while avoiding steric interference with the antigen-binding siteformed by the antibody domains.

Thus, in some embodiments, the fluorescent domain can include at least aportion of mRFP, mCitrine, mCerulean, GFP(wt), EBFP, Sapphire,T-Sapphire, ECFP, mCFP, CyPet, Midori-Ishi Cyan, mTFP1, EGFP, AcGFP,TurboGFP, Emerald, Azami Green, EYFP, Topaz, Venus, yPet, PhiYFP,mBanana, Kusabira Orange, mOrange, dTomato, DsRed-Monomer, mTangerine,mStrawberry, Jred, mCherry, HcRed1, mRasberry, or mPlum. Thus, whilereference is occasionally made herein to REDantibody, the use of thisparticular construct is merely an exemplary embodiment.

In some embodiments, the fluorescent domain can include an amino acidsequence that bears a specified level of amino acid sequence similarityto a reference polypeptide. The reference polypeptide may be, or be afluorescently active portion of, a fluorescent polypeptide.

Amino acid similarity of two polypeptides can be determined by aligningthe residues of the two polypeptides (for example, a candidatepolypeptide and the fluorescent domain of, for example, any one of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8) to optimize thenumber of identical amino acids along the lengths of their sequences;gaps in either or both sequences are permitted in making the alignmentin order to optimize the number of identical amino acids, although theamino acids in each sequence must nonetheless remain in their properorder. A candidate polypeptide is the polypeptide being compared to thereference polypeptide. In some embodiments, the reference polypeptidemay be, e.g., the fluorescent domain of any one of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, or SEQ ID NO:8 such as, for example, amino acids122-338 of SEQ ID NO:2, amino acids 3-219 of SEQ ID NO:4, amino acids3-235 of SEQ ID NO:6, or amino acids 3-235 of SEQ ID NO:8.

A pair-wise comparison analysis of amino acid sequences can be carriedout using the BESTFIT algorithm in the GCG package (version 10.2,Madison Wis.). Alternatively, polypeptides may be compared using theBlastp program of the BLAST 2 search algorithm, as described by Tatianaet al., (FEMS Microbiol Lett, 174, 247-250 (1999)), and available on theNational Center for Biotechnology Information (NCBI) website. Thedefault values for all BLAST 2 search parameters may be used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapx_dropoff=50, expect=10, wordsize=3, and filter on.

In the comparison of two amino acid sequences, structural similarity maybe referred to by percent “identity” or may be referred to by percent“similarity.” “Identity” refers to the presence of identical aminoacids. “Similarity” refers to the presence of not only identical aminoacids but also the presence of conservative substitutions. Aconservative substitution for an amino acid in a polypeptide of theinvention may be selected from other members of the class to which theamino acid belongs. For example, it is well-known in the art of proteinbiochemistry that an amino acid belonging to a grouping of amino acidshaving a particular size or characteristic (such as charge,hydrophobicity and hydrophilicity) can be substituted for another aminoacid without altering the activity of a protein, particularly in regionsof the protein that are not directly associated with biologicalactivity. For example, nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and tyrosine. Polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Conservative substitutions include, for example,Lys for Arg and vice versa to maintain a positive charge; Glu for Aspand vice versa to maintain a negative charge; Ser for Thr so that a free—OH is maintained; and Gln for Asn to maintain a free —NH2. Likewise,biologically active analogs of a polypeptide containing deletions oradditions of one or more contiguous or noncontiguous amino acids that donot eliminate a functional activity of the polypeptide are alsocontemplated.

The fluorescent domain can include a polypeptide with at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%amino acid sequence similarity to the reference amino acid sequence.

In certain embodiments, the fluorescent domain can include a polypeptidewith at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% amino acid sequence identity to the reference aminoacid sequence.

In some embodiments, at least one of the fluorescent domain, the firstantibody domain, or the second antibody can include an affinity tag.Affinity tags are routinely used to assist with the isolation and/orcollection of recombinant polypeptides. Affinity tags, their use, andthe methods of isolating polypeptides equipped with an affinity tag arewell known to those of skill in the art. Exemplary affinity tagsinclude, for example, a Histidine-rich tag (His-tag). A recombinantprotein containing a His-tag can be purified and detected easily becausethe string of histidine residues binds to several types of immobilizedmetal ions such as, for example, nickel, cobalt or copper, underspecific buffer conditions. In addition, anti-His-tag antibodies arecommercially available for use in assay methods involving His-taggedproteins. In either case, the tag provides a means of specificallypurifying or detecting the recombinant protein without aprotein-specific antibody or probe. It is also possible to usealternative conventional tags including, for example, tags that includethree or more amino acids, which bind to known corresponding affinityacceptors.

Another feature of the fluorescent fusion polypeptide involves theisoelectric point (pI). Table 2 summarizes the predicted pI values forexemplary antibody single chains with the predicted pI values forcorresponding antibody-RFP constructs. Generally, incorporating theantibody chain into a fluorescent fusion polypeptide lowers the pI.

TABLE 2 Molecular Weight Predicted pI B72.3 26.5 kDa 7.32 B72.3-RFP 51.5kDa 6.37 CA19.9 26.4 kDa 8.30 CA19.9-RFP 51.4 kDa 6.54 H23 26.2 kDa 9.03H23-RFP 51.2 kDa 7.37 4D5-8 27.0 kDa 8.32 4D5-8-RFP 45.1 kDa 6.19 CA12526.3 kDa 7.86 CA125-RFP 51.3 kDa 6.43

A compound can precipitate from solution when in an environment close toits pI. At physiological pH (approximately pH 7.4), the pI values ofmany single chain antibodies are close enough to the environmental pIthat they are at risk for precipitating out of solution. Incorporatingthe antibody chain into a fluorescent fusion polypeptide can alter thepI so that the antibody chain is less likely to precipitate atphysiological pH, thereby broadening the effective utility of theantibody chain. The V_(H) and V_(L) in the context of a whole antibodyhas a pI that is compatible with physiological pH. Once the V_(H) andV_(L) are engineered as single chain Fv, the pI can shift considerablyresulting in a pI close to the physiological pH, this would result inthe protein having a net zero charge and lead to aggregation andprecipitation. We observed 4D5-8 scFv with a (Gly₄Ser)₃ linker with apredicted pI of 8.32 to precipitate at pH 7.4. Whereas the correspondingREDantibody 4D5-8 with a predicted pI of 6.19 was soluble at pH 7.4.

One further feature of the fluorescent fusion polypeptides describedherein results from the polypeptides being able to be generated in situ.Because the polypeptides are genetically encoded, once a polynucleotideencoding the fluorescent fusion polypeptide is introduced into the cell,the cell will produce the polypeptide. Thus, intracellular targetmolecules can be detected without disrupting the cell to allow antibodyaccess to these target molecules.

In some embodiments, the fluorescent domain can include a photoactivatedfluorescent polypeptide. The mRFP1 (SEQ ID NO:4) is one such fluorescentpolypeptide. Photoactivated fluorescent polypeptides can be regeneratedby changing the light source. This can allow one to obtain repeatedsignal measurements from a single fluorescent fusion protein byregenerating the molecules ability to emit the fluorescent signal.

In another aspect, the invention provides compositions that include twoor more fluorescent fusion polypeptide described herein. The fluorescentdomain, first antibody domain, and second antibody domain of the secondfluorescent fusion polypeptide can, independent of the other domains, besimilar or different than the corresponding domain of any otherfluorescent fusion polypeptide in the composition. Thus, a compositionmay include a mixture of fluorescent fusion polypeptides that may havesimilar or identical antibody domains so that the two differentfluorescent fusion polypeptides bind to the same target molecules, butpossess different fluorescent domains so that the two differentfluorescent fusion polypeptides emit different fluorescent signals.Conversely, in other embodiments, a composition can include fluorescentfusion polypeptides that have different antibody domains so that the twofluorescent fusion polypeptides bind to different target molecules, buthave the same fluorescent domain so that they produce the samefluorescent signal. Finally, the composition can include two entirelydistinct fluorescent polypeptides so that the two fluorescent fusionpolypeptides in the composition bind to different target molecules andemit different fluorescent signals.

Different fluorescent signals may be characterized in any suitablemanner. For example, fluorescent signals may be characterized by thecolor of the signal emitted by the fluorescent domain. When moreprecision is desired, the fluorescent signal may be characterized interms of the wavelength of one or more characteristic emission peaks.

In another aspect, the invention provides polynucleotides that encodeany of the fluorescent fusion polypeptides described herein, and thecomplements of such polynucleotide sequences. Also included in thepresent invention are polynucleotides that hybridize, under standardhybridization conditions, to a polynucleotide that encodes any of thefluorescent fusion polypeptides described herein, and the complements ofsuch polynucleotide sequences. Also included in the present inventionare polynucleotides having a sequence identity of at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% to anucleotide sequence that encodes any of the fluorescent fusionpolypeptides described herein.

As used herein, “sequence identity” refers to the identity between twopolynucleotide sequences. Sequence identity is generally determined byaligning the residues of the two polynucleotides to optimize the numberof identical nucleotides along the lengths of their sequences; gaps ineither or both sequences are permitted in making the alignment in orderto optimize the number of shared nucleotides, although the nucleotidesin each sequence must nonetheless remain in their proper order. Acandidate sequence is the sequence being compared to a known sequence.For example, two polynucleotide sequences can be compared using theBlastn program of the BLAST 2 search algorithm, as described by Tatianaet al., FEMS Microbiol Lett., 1999; 174: 247-250, and available on theworld wide web at ncbi.nlm.nih.gov/BLAST/. The default values for allBLAST 2 search parameters may be used, including reward for match=1,penalty for mismatch=−2, open gap penalty=5, extension gap penalty=2,gap x_dropoff=50, expect=10, wordsize=11, and filter on.

In another aspect, the invention provides a method that includesintroducing a polynucleotide as just described into a cell. In someembodiments, the cell can be a prokaryote or a eukaryote. Thus, the cellmay be a single-celled organism such as, for example, a bacterium or ayeast cell. In other embodiments, the cell may be a cell from amulticellular organism. Exemplary cells from a multicellular organisminclude, for example, tumor cells. The method includes introducing thepolynucleotide into the cell using methods routine to those of skill inthe art and appropriate for introducing foreign nucleic acid moleculesinto the host cell. For example, routine heat shock transformationmethods may be used to introduce the polynucleotide into, for example, abacterial cell. For mammalian cells, the polynucleotide may beintroduced into the cells using, for example, nanoparticle delivery.

Thus, in yet another aspect, the invention provides a cell that includesa fluorescent fusion protein as described herein, a composition asdescribed herein that includes one or more fluorescent fusion proteins,or a polynucleotide that encodes a fluorescent fusion protein asdescribed herein. The cells may be in vitro—as may be the case with atumor cell obtained through a biopsy being analyzed—or in vivo—as may bethe case in methods, discussed in more detail below, in which thefluorescent fusion polypeptide is used to detect a particular cellpopulation in situ.

In some embodiments, the method can further include detecting afluorescent signal produced by the fluorescent domain of the fluorescentfusion polypeptide encoded by the polynucleotide and expressed by thehost cell. Depending upon the cell and the nature of the antibodydomains of the fluorescent fusion polypeptide encoded by thepolynucleotide, methods that include detecting the fluorescent signalcan have varying applications. For example, the polynucleotide may beintroduced into a microbe that is further genetically modified so that,after the genetically modified microbe is released into an environment,the movement or spread of the microbe in the environment may bemonitored. As such, the method can include detecting the fluorescentsignal over a plurality of time points so that time course data may beobtained. As another example, the cell may be a tumor cell. Theintroduction of a polynucleotide that encodes a fluorescent fusionpolypeptide may permit detection of tumor cells over time to, forexample, monitor the metastatic activity of the tumor cells.Alternatively, the polynucleotide may be introduced into tumor cells inadvance of surgery so that a surgeon is better able to distinguishdiseased and healthy tissues. This can help improve the likelihood thatall tumor cells are surgically removed as well as decrease the extent towhich healthy cells or tissues are removed. As yet another example, themethod can include non-invasive imaging; the fluorescent fusionpolypeptide may be detectable non-invasively through the skin. Forexample, the fluorescent fusion polypeptide may be designed to detectmarkers of inflammation and a polynucleotide encoding that polypeptideadministered to a subject. If the fluorescent domain emits a signal inthe near-IR portion of the spectrum, the signal may be visible throughthe skin.

In some embodiments, the fluorescent fusion polypeptide may providetherapeutic activity. Fluorescence can induce the conversion ofmolecular hydrogen (H₂) and molecular oxygen (O₂) to hydrogen peroxide:H₂+O₂→H₂O₂. With sufficient excitation (e.g., from white light),fluorescence from the fluorescent domain can produce enough H₂O₂ to killa cell harboring the fluorescent fusion polypeptide. This feature couldbe exploited in conjunction with, for example, illuminating tumor cells.Expression of the fluorescent fusion polypeptide may not only helpidentify diseased cells, but may also provide some level ofcytotoxicity.

In another aspect, one or more of the fluorescent fusion polypeptidesmay be used in any conventional detection method in place of an antibodywith corresponding antigen recognition activity. Indeed, use of afluorescent fusion polypeptide described herein may eliminate the finallabeling step of, for example, a conventional sandwich assay.

In yet another aspect the invention provides a kit that includes, inseparate containers, a first fluorescent fusion polypeptide and a secondfluorescent fusion polypeptide. In other embodiments, the kits caninclude, in separate containers, a first polynucleotide that encodes afirst fluorescent fusion polypeptide, and a second polynucleotide thatencodes a second fluorescent fusion polypeptide.

In another aspect, the invention provides a method of using thefluorescent fusion polypeptide as a nanoprobe. Although immunodetection,the mainstay of clinical diagnostic laboratories has changed formatsconsiderably over the past 50 years, the basic principles have remainedfundamentally the same: i.e., antibody-antigen interactiondetermination, either qualitative (yes/no), if yes, where (imaging), orquantitative (how much), and various combinations of these metrics. Thescope for the application of immunoassays is wide, from over-the-counterpregnancy tests, HIV testing, substance of abuse monitoring, detectingmycotoxins in food and feed, measuring markers of cardiac health toscreening biopsies for tumor-associated markers. The ability to readilymanipulate antibody gene sequences using simple bacterial expressionsystems allowed various strategies to be developed for accessing andmodifying antibodies. One application of such modified antibodiesinvolves creating recombinant antibody fragments as fusions with markermolecules. Currently, it is possible to tag antibodies with differentcolored fluorescent chemical tags and to use these reagents inmulti-analyte detection. However, antibodies tagged in this manner canvary from batch to batch in the amount of fluorescent chemical that isconjugated to the antibody. That is, the amount of fluorescent chemicaltagged to the antibody is not absolute, some may have more than othersand in some instances the chemical coupling can interfere with thebinding properties of the antibody. Hence, use of these taggedantibodies yields an ‘averaged’ result, which is acceptable for aqualitative assay, but is limited in quantitative assays.

In contrast, the fluorescent fusion polypeptides described hereininclude a single fluorophore so that every labeled antibody carries asingle fluorescent molecule to tag antigens with a particular color.These fluorescent fusion polypeptides make possible new approaches basedon the integration of multiple readouts in a range of quantitativeand/or qualitative settings, from cell surfaces to antigens inmicroarrays, etc.

We have engineered monomeric red fluorescent protein mRFP from a coralDiscosoma (Campbell, R. E., et al., 2002, Proc Natl Acad Sci USA 99,7877-7882.) and inserted it between the V_(H) and V_(L) domains of arange of recombinant anti-cancer antibodies such as, for example, B72.3,CA19.9, and 4D5-8 replacing the flexible peptide linker. This is,however, a general approach that may be used to produce fluorescentfusion polypeptides that include a portion of V_(H) and V_(L) domains ofany antibody of interest. Indeed, we have applied this approachsuccessfully to six different scF_(v)s. Moreover, the mRFP can bereadily interchanged with a range of other fluorescent proteins thathave almost identical external tertiary structure (Tsien, R. Y., 2009,Angew Chem Int Ed Engl 48, 5612-5626), thus opening up the possibilityof creating palettes of stable recombinant monoclonal antibodies withdefined spectral properties for use in, for example, protein arrays,live cell imaging, and/or immunocytochemical imaging.

As noted above, in some applications, the fluorescent fusionpolypeptides may be used for in vivo imaging, using the fluorescentfusion polypeptides to label, for example, diseased cells and/or tissuesto aid surgery. The fluorescent fusion polypeptides may provide anavigational map to assist surgeons in identifying both diseased andhealthy tissue on a molecular level and, therefore, assist in increasingthe accuracy and precision of the surgical procedure to remove diseasedtissue while limiting the extent to which healthy tissue is collaterallyremoved during surgery.

In the case of cancer, surgery with molecular fluorescence imaging usingactivatable cell-penetrating peptides decreased the residual cancer andimproved survival in a mouse xenograft model (Nguyen, Q. T., et al.,Proc Natl Acad Sci USA 107, 4317-4322). Thus, fluorescence imaging mayprovide a dual opportunity to exploit the fluorescent fusionpolypeptides described herein. The fluorescent fusion polypeptides maybe useful for detecting the presence of tumors using in vitro detectionmethods on cells or tissues obtained from a biopsy sample, then used invivo for guiding the subsequent surgical removal or ablation of diseasedcells and/or tissues.

In yet another aspect, fluorescent antibodies may be used in connectionwith pest management and/or disease control. Evolving methods forcontrol of vector-borne diseases rely on modification of insects ratherthan elimination of insects by, for example, application of pesticides.These new strategies involve either direct transformation of an insectgenome via mobile DNA elements (transgenesis) or expression of geneproducts in the host insect via transformed symbiotic microbes(paratransgenesis). Transgenesis and/or paratransgenesis may supplementconventional insect control methods and, therefore, decrease the extentto which conventional methods must be used—i.e., transgenesis andparatransgenesis may not necessarily completely supplant traditionalinsecticide campaigns but can decrease reliance on insecticides and,therefore, the negative environmental effects of insecticide use.

Paratransgenesis is a “Trojan Horse” approach to control of diseasetransmission. It employs the interactions between disease-transmittingvectors, bacterial symbionts of the vectors, and transmitted pathogens.Symbiotic bacteria are isolated and genetically transformed in vitro toexport molecules that interfere with pathogen transmission. Thegenetically altered symbionts are then introduced into the host vectorwhere expression of engineered molecules affects the host's ability totransmit the pathogen, i.e., its vector competence. This approachattempts to decrease pathogen transmission without adverse effects onthe vectors themselves. Furthermore, it can employ, as a gene deliverymechanism, bacterial flora native to the host vector. There are severalrequirements for such an approach to work: (1) A population of symbioticbacteria must exist within a given disease-transmitting vector, (2)Symbiotic bacteria should be specific to a given vector, (3) Bacterialsymbionts should be amenable to culture and genetic manipulation, (4)Genetically altered symbionts should remain stable, (5) Fitness of thegenetically altered symbionts to re-infect host vectors should not becompromised. Furthermore, their normal symbiotic functions should not bealtered, (6) Transgene products released from the genetically alteredsymbionts should interact with the target pathogen(s) and (7) A methodmust exist for dispersal of the genetically altered symbionts amongstnaturally occurring populations of vectors with minimal non-targetspread of foreign genes to environmental bacteria and other arthropods.

So, for example, Pierce's Disease is a deadly disease of grapevines thatcauses significant economic loss to the wine industry of California. Itis caused by the bacterium Xylella fastidiosa, which is spread byxylem-feeding sharpshooters. The predominant vector of this disease inthe U.S. is the Glassy Winged Sharpshooter (GWSS), Homalodiscavitripennis. Xylella is xylem-limited in host plants and is not thoughtto survive outside the vector insect or host plant. GWSS is axylem-feeder and carpets of bacteria on the surface of the precibarialchamber of the mouthparts of the insect have long been thought to be theprinciple source of transmission of pathogenic Xylella strains intosusceptible plants. The anterior mouthparts of H. vitripennis arecolonized with a variety of other environmental bacteria some of whichappear to have a symbiotic association with the insect. Two suchorganisms are Alcaligenes xylosoxidans dentrificans (AXD) and Pantoeaagglomerans, both environmental gram-negative bacteria commonly found inthe rhizosphere of grape plants. Both X. fastidiosa and the twosymbionts are located in physical proximity in the cibarium of GWSS inan extracellular environment. Fluorescent antibodies as described hereinmay be engineered into AXD and/or P. agglomerans so that the fluorescentantibodies are expressed, target the surface of Xylella, and preventtransmission of this pathogen by H. vitripennis. The fluorescentantibodies may provide two related but distinct functions: they can (1)function as transmission-blocking molecules that disrupt Xylellapathogenesis in grape plants, and (2) intrinsically fluoresce so thatone can monitor gene flow in the environment as a risk assessmentstrategy.

To develop a panel of anti-X. fastidiosa surface proteins, five scFvlibraries were constructed using spleens from mice previously immunizedwith whole, heat-inactivated X. fastidiosa cells. These libraries wereused to select and isolate scFv's against the surface molecule MopB(4XfMopB1 & 4XfMopB3) of X. fastidiosa. Antibody scFv's selected againstsurface components of X. fastidiosa can be engineered into thefluorescent antibody format described herein and evaluated forplasmid-based expression in E. coli and P. agglomerans. Candidates thatare expressed and secreted in P. agglomerans can be used to establishstable bacteria that can express the antibody using transposon insertioninto the P. agglomerans genome.

On the outer membrane of Gram-negative bacteria, macromolecules such aslipopolysaccharides and proteins are produced. MopB is the major outermembrane protein in X. fastidiosa. MopB is a member of ompA proteinfamily. Two constructs of X. fastidiosa MopB (truncated and full lengthmature) have been expressed and purified.

Furthermore, mouse immunoglobulin DNA libraries against heat-killedwhole X. fastidiosa have been constructed and used in ribosometechnology to select specific scFv antibodies against MopB and thesehave been characterized to confirm binding to X. fastidiosa. Using theselibraries, one can isolate scFv antibodies against other surfaceproteins that may play a critical role in X. fastidiosa attachment orpathogenicity in the insect or plant. Since the complete genome of X.fastidiosa is known, using a bioinformatics approach can identifyadditional potential X. fastidiosa surface proteins. Thus, one can clonethese candidates in a similar manner as MopB and use the recombinantproteins to isolate alternative surface specific scFv's.

In other approaches, paratransgenesis using a fluorescent polypeptide asdescribed herein may be used to inhibit the effects of pathogensaffecting other agricultural species, maricultural species, or livestockspecies. With the platform established as just described, analogouscorresponding paratransgenesis systems may be designed. Such systems canpermit inhibition of the pathogen and/or detection of the engineeredmicrobe.

In another aspect, microbes may be genetically-engineered to produce andexport fluorescent antibodies that interfere with animal pathogens. Forexample, E. coli Nissle (EcN) may be used as a bacterial delivery systemto produce and export fluorescent antibodies that interfere withpathogenesis of, for example, Clostridium difficile. In this aspect, EcNare used for paratransgenesis. Resident bacteria such as, for example,EcN are isolated and genetically transformed in vitro to exportmolecules that interfere with pathogen transmission. The geneticallyaltered bacteria are then introduced into the host where expression ofengineered molecules disrupts virulence of the pathogen such as, forexample, C. difficile. This approach results in a decrease ofdeleterious effects of the pathogen by delivering local passiveimmunity. Furthermore, it employs, as a gene delivery mechanism,bacterial flora that can reside in the host. This paratransgenicplatform can be used to produce recombinant EcN that expressesantibodies that specifically bind C. difficile Toxin A and/or Toxin B.

Molecular Design

The X-ray crystallographic structure of Fab B72.3 (PDB: 1BBJ) revealsthat the distance between the C-terminus Ser114 on the variable heavychain and the N-terminus Asp1 on the variable light chain isapproximately 34 Å. A search of the Protein Data Base (PDB) was carriedout to identify structures with N- and C-termini in the same spatialplane with spacing close to 20-30 Å. Fortuitously, mRFP met thesecriteria with the distance between Va17 and His221 of the termini infast maturing red fluorescent protein DsRed variant (PDB 2VAE) beingapproximately 26 Å. Thus, direct fusion of the V_(H) to the N-terminusand V_(L) to the C-terminus would have resulted in a linkage 8 Å shorterthan the optimal spacing revealed by X-ray crystallographic structure ofB72.3. This was overcome by modeling the N- and C-termini to includeGly₄Ser linkers on each end of RFP (FIG. 1). The resulting model couldaccommodate the variable regions of the antibody with a moleculargeometry approaching that determined from the X-ray studies.

Molecular Biology

The V_(H)-RFP-V_(L)-His-Tag (REDantibody) constructs were assembled in amodified pET-26b vector (EMD Chemicals Inc., Gibbstown, N.J.) with apelB leader sequence to direct secretion to the periplasm of E. coli(FIG. 2A) or in a modified pET-32a vector (EMD Chemicals Inc.,Gibbstown, N.J.) without a pelB leader for cytoplasmic expression. Thesynthetic genes of the V_(H)-V_(L) scFv antibodies were designed toencode an in-frame BamHI site between the V_(H) and V_(L) regions andthe whole flanked by NcoI and NotI sites for in-frame directionalcloning. The mRFP gene was inserted into each of these plasmids to makeexpression plasmids pBAK1B72.3RFP and pBAK1CA19.9RFP as shown in FIG.2A. The pBAK14D5RFP was assembled in a similar manner after removing aninternal BamHI site. The expression cassettes starts from pelB leadersequence followed by V_(H) chain, RFP, V_(L) chain and an octa-His-tagat the C terminus of the resulting protein sequence as shown in FIG. 2B.

Protein Expression & Purification

The recombinant scFv and the corresponding REDantibody proteins wereexpressed in BL 21 (DE3) pRARE E. coli strain and recovered from theperiplasmic extract or in Rosetta gami B(DE3) E. coli strain andrecovered from the cytoplasmic extract and purified via Ni-NTA affinitychromatography. Protein expression and purification processes weremonitored by SDS-PAGE. The protein concentration of the recoveredfunctional protein was determined for each construct: mRFP (20 mg/L),REDantibody 4D5 (5 mg/L), REDantibody CA19.9 (0.88 mg/L), andREDantibody 72.3 (0.9 mg/L). The predicted molecular weights of the scFvand REDantibody recombinant proteins were approximately 25 kDa and 51kDa, respectively, as shown in FIG. 3. The SDS-PAGE analysis of theNi-NTA affinity enriched recombinant antibodies also had other proteinbands corresponding to co-enriched E. coli host proteins. The sizeexclusion column was calibrated using a standard range 200 kDa-12 kDa(Sigma-Aldrich, St. Louis, Mo.) prior to use (FIG. 4A). The mRFP proteinelution corresponded to monomer of 25 kDa (FIG. 4B). A singleREDantibody peak eluted at fractions/time corresponding to ˜51 kDa,(FIG. 4C). Moreover, the color of REDantibody E. coli culture,periplasmic, and elution fractions were pink (FIG. 5 A-C, respectively).

Fluorophor Measurements

The excitation and emission peaks of REDantibody 4D5-8 were determinedto be 584 nm and 607 nm as shown in FIG. 15A. The concentrationdependent emission at 607 nm permitted accurate detection of 9.5 pmoleof this REDantibody as shown in FIG. 15B.

BIAcore Measurements

The association and disassociation rate for REDantibody 4D5-8 binding top185HER2-ECD was determined to be 2.19×10⁻⁴±0.13×10⁻⁴ M⁻¹s⁻¹ and4.43×10⁻⁵±1.04×10⁻⁵ s⁻¹, respectively and the KD calculated to be2.2±0.8 nM.

Immunofluorescent Staining and Confocal Microscopy

Functional analysis of the REDantibody was based on well-characterizedproperties of B72.3 and CA19.9 antibodies to recognize sialyl-Tn andsialylated Lewis (Le)^(a) blood group antigen, respectively, which arepart of a panel of markers used in cancer diagnostics. The samesialyl-Tn antigen has previously been detected on the surface of thehuman pathogen Trypanosoma cruzi using B72.3 monoclonal antibody.

The CA19.9 also binds to sialyl glycans on the parasite surface. The 4D5REDantibody was constructed for use as a negative control since itrecognizes a peptide epitope on p185HER2, but not sialyl glycan. Thiswas confirmed by the fluorescent staining of T. cruzi epimastigotesusing purified recombinant anti-glycan REDantibody shown in FIG. 6A andFIG. 6B. The control REDantibody 4D5 did not label the parasites (FIG.6C). The maximum intensity fluorescence staining image with REDantibodyCA19.9 (FIG. 6D) revealed the appearance of a cleft area on the parasitesurface with reduced staining Confocal three-dimensional imaging (FIG.6E) confirmed the presence of a depression corresponding to the cleftobserved earlier. The cut away image of the confocal image revealed thatthe staining was restricted to the surface of the parasite (FIG. 6F).

Immunofluorescent tagging of molecules, in particular antibodies, is awidely used tool in clinical diagnostics and research. Historically,fluorophores exemplified by FITC, TRITC/CY-3, and TRITC/CY-5 have beenchemically linked to antibodies, resulting in some instances withreduced binding to target antigen and/or a heterogeneous number oflabels incorporated into each antibody molecule. The heterogeneity oflabels incorporated into each antibody molecule limits the utility ofthese molecules for quantitative analysis. Significant advances havebeen made in conjugation chemistry to attempt to overcome some of thesedifficulties. For example, approaches have been developed that result inrecombinant scFv that incorporate a free cysteine at the C-terminus forspecific conjugation chemistry. Others have gone one step further andfused scFv directly to GFP. However conventional scFv are prone toaggregation. It is not clear whether fusing a scFv directly to GFP wouldimpact the aggregation properties of the scFv. More recently, GFP hasbeen modified to incorporate two loops that can form recognition motifseven though the resulting molecule is not an antibody as such.

We sought to overcome two issues faced with creating antibodyfluorophores. Firstly, we incorporated a single fluorophore per bindingsite. Secondly, we spatially orientated the V_(H)/V_(L) interfaces foroptimal pairing. Our earlier attempts to construct similar bridgedmolecules using a related β-barrel structure of green fluorescentprotein (GFP) resulted in molecules that did not bind to the targetantigen.

The vectors constructed encoding the REDantibodies are based on the pETseries as shown in FIG. 2. The use of the T7 promoter and the BL21(DE3)host results in high level of expression of the recombinant protein, themajority of which is retained within the cell cytoplasm despite having aPelB leader sequence to direct the polypeptide into the periplasm. Theamount of REDantibody recovered varied for each antibody construct. 4D5had the highest recovery (5 mg/L) whereas the recovery of thecorresponding anti-sialyl glycan antibodies were both just below 1 mg/L.Both levels are comparable to the recovery of scFv from E. coli. Therecovery of anti-sialyl glycan REDantibodies may have been reduced sincethe secreted, correctly folded, and functional REDantibody in both casesbound to sialic acid, which is present on the host bacterial surface.

Sufficient REDantibody was recovered from the periplasmic extract topermit further affinity purification and functional analysis. TheSDS-PAGE of the affinity isolated CA19.9 scFv and the correspondingREDantibody had proteins of 25 kDa and 51 kDa, respectively, as shown inFIG. 3A and FIG. 3B, respectively. The REDantibody also had a doubletband at 26 kDa, which could be due to cleavage within the mRFP thatoccurs when the sample is boiled in SDS as observed with DsRed.

Size exclusion chromatography on the affinity purified proteins was usedto determine whether the molecules were monomeric or multimeric. Each ofthe mRFP and REDantibody eluted with a single peak, corresponding to 25kDa and 51 kDa, respectively, as shown in FIG. 4B and FIG. 4C,respectively. These data indicate that both proteins are monomeric.

Features of this REDantibody platform include the intrinsic color of thebacteria producing the active molecule and the intrinsic color of thepurified proteins. Consequently, sophisticated detection in thepurification steps is not required. The excitation wavelength ofREDantibody is 584 nm and the emission wavelength is 607 nm, which areidentical to mRFP (Campbell et al., 2002 Proc Natl Acad Sci USA99:7877-82; Khrameeva et al., 2008 Biochemistry (Mosc) 73:1085-95)confirming that the fusing immunoglobulin domains to both ends of afluorescent protein does not compromise the fluorescent properties.Moreover, it may be possible to detect as little as 9.5 pmoles ofREDantibody using a standard fluorometer as shown in FIG. 15B. This maypermit quantitative analysis of target antigen. The kinetics of bindingfor the anti-glycan REDantibodies were difficult to perform since adefined antigen preparation was not readily available. Thus we carriedout the binding studies on the REDantibody 4D5-8. The calculated K_(D)value for REDantibody 4D5-8 of 2.19 nM was within the values determinedindependently for the 4D5-8 scFv with a V_(H)-(Gly4 Ser)3 linker V_(L)orientation of 194 pM (Worn and Pluckthun, 1998 FEBS Lett 427:357-61),and 9.4 nM for HERCEPTIN (Genentech, Inc., South San Francisco, Calif.)(Troise et al., 2008 FEBS J 275:4967-79), further confirming that thebinding characteristics are not compromised in this format.

These characteristics of the REDantibody permit its use with otherconventional dyes for FACs analysis, immunochemistry, and confocalmicroscopy that utilize a range up to 543 nm. In this study we used theREDantibody B72.3 and CA19.9 to visualize the carbohydrate antigenssialylTn and sialylated Lewis (Le)a on the surface of T. cruziepimastigotes, respectively, using immunofluorescence (FIG. 6A, FIG. 6Band FIG. 6D) and confocal (FIG. 6E and FIG. 6F) microscopy. The bindingof the REDantibody CA19.9 is restricted to the surface of the parasite(FIG. 6B). The negative control REDantibody 4D5-8 did not label theparasites when examined using immunofluorescence microscopy, implyingthe specificity of binding observed using the B72.3 and CA19.9antibodies was introduced by the antibody binding domains (FIG. 6C).

One feature of the REDantibodies described herein include robuststability; the slides prepared for this study could be viewed for atleast a week without noticeable loss of signal, indicating robuststability of the REDantibody. Another feature is that the monomeric redfluorescent protein (mRFP1) derived from DsRed is stable within a widerange of pH 5.0-12.0 and in 6M urea. Thus, the antibody/antigeninteraction may be disrupted by adjusting the pH and/or adding urea, andthe released fluorophore may be quantified in solution. Yet anotherfeature is that the REDantibody molecule is photo activated. Thus, theREDantibodies described herein permit detecting a target with increasedsensitivity through the use of, for example, time-resolved dual laserpulsed analysis. Yet another feature is that the REDantibody cangenerate reactive oxygen species (e.g., H₂O₂) upon exposure to whitelight. This may potentially provide a wide range of therapeuticapplications such as, for example, photo-ablation of target cells.Additionally, with a far red emission spectra, these types of moleculemay have utility in in vivo whole body imaging.

Assembling the REDantibody using the fluorophore as a bridge introducescertain features. Firstly, with an integrated fluorophore, a singlereagent has been created, which reduces time and cost and increasesreproducibility of the binding assay. Secondly, the antibody bindingsite and fluorophore are stoichiometric. Thus, the signal generated isdirectly proportional to the amount of antibody bound (i.e.,quantitative). Finally, expression and purification steps can bemonitored without the need for the expensive equipment since thebacteria and the recombinant proteins are visible with a distinct redcolor.

The platform may be applied using other suitable fluorescent proteindomains. Thus, the fluorescent domain can include at least a portion ofmRFP, mCitrine, mCerulean, GFP(wt), EBFP, Sapphire, T-Sapphire, ECFP,mCFP, CyPet, Midori-Ishi Cyan, mTFP1, EGFP, AcGFP, TurboGFP, Emerald,Azami Green, EYFP, Topaz, Venus, yPet, PhiYFP, mBanana, Kusabira Orange,mOrange, dTomato, DsRed-Monomer, mTangerine, mStrawberry, Jred, mCherry,HcRed1, mRasberry, or mPlum. Thus, the preceding description of theconstruction of the REDantibody is merely exemplary; analogousconstructs may be designed using any suitable fluorescent proteindomain.

Moreover, this platform may be applied to other existing mAbs to createthe next generation of diagnostic and therapeutic molecules. Theplatform may also be used to create libraries of V_(H) and V_(L) domainsthat may be readily accessed using high throughput non-isotopicscreening and not rely on phage or other types of display selectiontechnologies. In general, the use of other pre-folded protein domainswith N- and C-termini in the same spatial plane with spacing close to20-30 Å may be used in this modular approach to antibody engineering.Furthermore, the monomeric fluorophore also may be engineered to alterthe excitation/emission spectra, thus enabling multicoloredantibody-based reagents for multi-analyte detection or co-localizationstudies.

We engineered a scFv 4D5-8 fusion with mRFP1 based on the X-ray crystalstructure of 4D5-8 Fab (PDB 1N8Z; Cho et al., 2003 Nature421(6924):756-760) and the modeled structure of mRFP1 based on the DsReddimer (PDB 1G7K; Yarbrough et al., 2001 Proc Natl Acad Sci USA98(2):462-467). We fused the C-terminus of 4D5-8 V_(H) chain to theN-terminus of mRFP1 and fused the 4D5-8 N-terminus of V_(L) to theC-terminus of mRFP1, with the addition of optimal linkers separatingboth protein functions yet preserving the correct spatial dimensions forV_(H)/V_(L) interaction and fluorophore activity (FIG. 8A). With theaddition of five amino acid (Gly4Ser) linkers to either end of themRFP1, the N-terminus, C-terminus, or both can readily be extended tocreate a distance of, for example, approximately 35 Å between the mRFP1domain of the fusion and the V_(H)/V_(L) chains. The predicted structureshows that the mRFP1 does not interfere with the scFv binding site,which is fully available to contact the target ligand. This spatialgeometry permits the docking of mRFP1, or any of its derivatives,between the antibody V_(H)/V_(L) or V_(L)/V_(H) chains resulting infunctional scFv-fluorophore fusions.

Construction of the Expression p4D5-8mRFP1 Vector.

Construction of an exemplary vector expressing an exemplaryscFv-fluorophore fusion is shown in FIG. 8B. A 714 bp NcoI/NotI fragmentencoding the 4D5-8 scFv V_(H)-V_(L) orientation with a five amino acid(Gly4Ser) linker incorporating an in-frame BamHI (GGA TCC) restrictionsite (encoding amino acids Gly Ser) was inserted into a modified pET-26bvector. Modified mRFP1 with similar (Gly4Ser) linkers on both ends wascloned into the scFv BamHI site.

Protein Expression and Purification.

The 52 kDa protein (4D5-8 V_(H)-RFP-V_(L)) was expressed in E. coli BL21(DE3) pRARE or Rosetta gami B(DE3). During the growth and expressionphase the bacteria had a distinct red pigmentation. The protein wasinitially enriched by immobilized metal ion affinity chromatography(IMAC) and fractions analyzed by SDS-PAGE (FIG. 9a ) and western blot(FIG. 9b ), and then analyzed by gel filtration chromatography comparedwith mRFP1 and standards confirming the basically monomeric state (FIG.9c ). The fractions with the red pigment corresponded to fractions32-37. The purified monomeric protein was further analyzed by SDS-PAGE(FIG. 9d ). Western blot analysis of the eluted fractions (FIG. 9b ),revealed a band above the expected size, and smaller products. Thehigher band probably corresponds to the protein with the PelB leaderintact, recovered from the cell lysis and the lower bands are thebreakdown products due heat induced proteolysis of mRFP1 at thechromophore site which has also been reported by others (Serebrovskayaet al., 2009 Proc Natl Acad Sci USA 106(23):9221-9225). The proteolysisoccurs upon sample preparation for SDS-PAGE. A final yield of the 4D5-8REDantibody was 5 mg/L of bacterial culture.

Flow Cytometry and Fluorescence Microscopy Analysis.

To further characterize the binding activity of 4D5-8 REDantibody weused flow cytometry and fluorescence microscopy. As shown in FIG. 10,purified red antibody effectively recognized SKBr-3 breast carcinomacells that are characterized by high expression levels of p185HER-2-ECD(FIG. 10a ) and is used as +++ positive control in the FDA approvedHercepTest (Dako North America, Inc., Carpinteria, Calif.) and did notrecognize the MDA-MD-231 cancer cells (FIG. 10b ) which do not expressp185HER-2-ECD and are used as a zero negative control in the same test(Prichard et al., 2008 Clin Lab Med 28(2):207-222, vi).Immunofluorescent staining of SKBr-3 breast cancer cells revealed thatpurified 4D5-8 REDantibody effectively accumulates on the surface ofthese cells after a 30-minute incubation at ambient temperature as shownin FIG. 10c and FIG. 10 d.

Few genetically encoded fluorescent antibodies have been reported todate (Griep et al., 1999 J Immunol Methods 230(1-2):121-130; Lu et al.,2005 J Zhejiang Univ Sci B 6(8):832-837; Serebrovskaya et al., 2009 ProcNatl Acad Sci USA 106(23):9221-9225), in part because it has been provendifficult to maintain native affinity activity of antibody domains andmaintain native fluorophore activity of the fluorophore domain. Effortshave included omitting the antibody component and creating a GFP-basedbiosensor possessing antibody-like properties using the GFP as ascaffold and introducing two proximal binding loops (Pavoor et al., 2009Proc Natl Acad Sci USA 106(29):11895-11900). Conventional antibodies,however, have six proximal binding loops that, combined with subtlevariations in the framework regions, can provide a much greater range ofpossible affinities and specificities. On the other hand, the rigidβ-barrel structure of fluorescent proteins impose limits on the numberof changes that can be accommodated without altering the spectralproperties (Abedi et al., 1998 Nucleic Acids Res 26(2):623-630).

Ideally, a fluorescent antibody can possess as much nativeligand-binding activity as possible from its antibody domains and asmuch of the native fluorophore spectral properties as possible from itsfluorophore domain.

One way to retain as much ligand-binding activity and as muchfluorophore activity as possible can involve a modular approach wherethe two functionalities reside in distinct, non-overlapping regions of asingle molecule. However, recombinant scFv with linkers can be prone todisassociation and aggregation (Worn and Pluckthun, 2001 J Mol Biol305(5):989-1010). Yet, in the context of a Fab molecule, the V_(H)/V_(L)remain associated. Nature provides two solutions for stabilizingV_(H)/V_(L) pairs. In conventional antibodies, the CH1 and the constantlight chain orient and hold the V_(H) and the V_(L) in place for optimalinterface pairing. In the other case, the V_(L) is not required and onlythe V_(H) is required for ligand binding, as is the case in camelantibodies (Hamers-Casterman et al., 1993 Nature 363(6428):446-448).Although making direct fluorophore fusions with engineered V_(H) domainsis possible, the vast majority of characterized monoclonal antibodiesconsist of both V_(H) and V_(L) domains. Thus, an approach that couldconserve the Fab-like V_(H)/V_(L) pairing and permit fusion with afluorescent protein can produce a fluorescent antibody that possessesreliable spectral properties and high affinity and specificitycharacteristics.

We have devised a novel modular approach that produces efficientgeneration of a stable genetically-encoded intrinsically labeledfluorescent antibody. We have inserted the β-barrel structure of thefluorophore between the variable domains. These modular constructs notonly introduce the fluorophore into the fusion molecule, but alsostabilizes the positional orientation of the variable domain interfaces,resulting in Fab-like V_(H)/V_(L) pairing and, therefore, Fab-likeligand binding.

In the embodiment described in Example 1, the V_(H) C-terminus is fusedto the N-terminus of mRFP1, replacing the CH1 domain. The C-terminus ofthe mRFP1 is fused to the N-terminus of the V_(L), providing analternative anchor for the V_(L) and thus eliminating a need for the CL.The exemplary 4D5-8 REDantibody described in Example 1 has the followingcharacteristics: it is readily expressed and isolated from E. coli, witha molecular weight similar to the Fab fragment; the fluorophore activityremains unaltered; it is monomeric; it is stable; it retains thespecificity of the parental antibody; and it is simple to use as asingle reagent in immunohistochemistry, flow cytometry, and/or molecularimaging.

The 4D5-8 REDantibody molecule may be used, for example, for thedevelopment of a diagnostic test for Her2-positive breast cancer inbiopsy samples by immunohistochemistry or possibly for circulatingcancer cells by FACs. Using this platform it should be possible tocreate panels of REDantibodies against a range of cancer and otherbiomarkers. Moreover, since the palette of monomeric fluorescentproteins (mHoneydew to mPlum) are based on the basic architecture ofmRFP1 used in this study, it should be possible to exchange the redfluorophore for these other colored proteins that have similar 11β-sheet barrel-like structure (Zimmer, 2002 Chem Rev 102(3):759-781).The combination of different antibody specificities each with a distinctcolor spectrum opens up the possibility of antibody mediated multiplexedanalysis by fluorescence microscopy and FACs analysis. Exemplary modularfluorescent antibodies that produce colors other than the red colorproduces by the 4D5-8 REDantibody are described in Example 3.

Recently, p185HER-2-ECD expressing cancer cells have been targeted using4D5 scFv fused to the N-termini of KillerRed (dimeric) reporter(Serebrovskaya et al., 2009 Proc Natl Acad Sci USA 106(23):9221-9225).Upon light irradiation in oxygen rich environment the KillerRed produceshighly reactive oxygen species (ROS) which oxidise molecules in closeproximity resulting in p185HER-2-ECD expressing cancer cell damage anddeath (Serebrovskaya et al., 2009 Proc Natl Acad Sci USA106(23):9221-9225). These properties have also been assigned to otherfluorescent proteins and it has been experimentally confirmed thatduring formation of the chromophore one molecule of H2O2 is generatedfor each molecule of fluorescent protein (Zhang et al., 2006 J Am ChemSoc 128(14):4766-4772). Therefore the 4D5-8mRFP1 described could alsogenerates ROS upon exposure to ambient light, thus it may also havetherapeutic potential in targeted cell ablation and photodynamictherapy. Another advantage of mRFP1 is its far-red spectrum which has agreater tissue penetration permitting use for in vivo imaging (Yurchenkoet al., 2007 Transgenic Res 16(1):29-40). Antibody targeted mRFP1 mayalso be evaluated for use in vivo in diagnostic and/or therapeuticapplications. This dual modular assembly permits improvements to beincorporated on the scFv and the fluorophore, resulting in opportunitiesfor further combinatorial optimised binding and more desirable spectralproperties. Similar REDantibody constructs based on the anti-sialyl-Tnand anti-sialylated Lewis (Le)a antibody B72.3 (Brady et al., 1991 J MolBiol 219(4):603-604) and CA19.9 (Koprowski et al., 1979 Somatic CellGenet 5(6):957-971) respectively have also been assembled.

Assembling the REDantibody using the genetically encoded fluorophore asa bridge results in a stabilized, stoichiometric molecule with eachbinding site having a single reporter molecule, allowing quantitativeanalysis. This platform may also be applied to other existing mAbs tocreate the next generation of diagnostic imaging and sorting molecules.The modular platform may also be used to combine natural and syntheticimmunoglobulin V_(H) and V_(L) domains with libraries of fluorophoresbased on mRFP1 and GFP to create a palette of highly specific coloredbinding molecules that retain optimal binding and spectral properties.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1

Materials and Methods

Molecular Design and Visualization

Structure of B72.3 and 4D5 antibodies were downloaded from PDB database(PBD: 1BBJ and 1FVC respectively). RFP structure was predicted usingSwiss-Model Workspace server. Further modeling was performed usingMIFit+ software version 2009.09-1 (Rigaku Americas Corp., The Woodlands,Tex.) and protein models were viewed using PyMOL software version 1.1(DeLano Scientific LLC, Palo Alto, Calif.).

Plasmids, Primers and Synthetic DNA

Plasmid pBAK1 and pA were previously constructed in our laboratory isbased on pET-26b and pET-32a vectors, respectively (EMD Chemicals Inc.,Gibbstown, N.J.). All primers were purchased from Invitrogen Corp.(Carlsbad, Calif.). Synthetic DNA sequences of B72.3 and CA19.9 antibodyvariable domains in V_(H)-V_(L) orientation were codon optimized for E.coli expression and purchased from Epoch Biolabs, Inc. (Missouri City,Tex.) as plasmids pBSK-B72.3 and pBSK-CA19.9 respectively, with NcoI,BamHI and NotI restriction sites to facilitate construction of theexpression vectors. Plasmid pAK19 encoding the 4D5 V_(H) and V_(L) wasprovided by Dan Yansura (Genentech, Inc., South San Francisco, Calif.).Plasmids pMTRFP and pMTBFP, with mRFP1 and BFP genes, respectively, werea gift from Professor Ray St. Ledger (University of Maryland, CollegePark, Md.). Synthetic DNA sequences of mCitrine and Cerulean were codonoptimized for E. coli expression and purchased from Epoch Biolabs, Inc.(Missouri City, Tex.) as plasmids pBSK-mCit and pBSK-Cer respectively,with flanking BamHI restriction sites to facilitate insertion into theexpression vectors.

Bacterial Strains, Growth Media and Recombinant DNA Technique

XL1-Blue Escherichia coil strain (Stratagene, Agilent Technologies,Inc., Santa Clara, Calif.) was used for plasmid construction steps. Toexpress recombinant antibodies BL21 (DE3) or Rosetta gami B(DE3) strainof E. coli (EMD Chemicals Inc., Gibbstown, N.J.) were used. E. colicells were grown in Lysogeny Broth (LB) (Bertani, 2004) or LB agarplates. Kanamycin sulfate, carbenicillin, and chloramphenicol were usedat 30 μg/mL, 100 μg/mL, and 34 μg/mL final concentration, respectively.Plasmid DNA was isolated using QIAprep Spin Miniprep Kit (QiagenInc.—USA, Valencia, Calif.) and DNA from gel was purified using QIAquickGel Extraction Kit (Qiagen Inc.—USA, Valencia, Calif.). Escherichia colicells were transformed using standard heat shock methods. Restrictionand modification enzymes were purchased from New England Biolabs, Inc.(Ipswich, Mass.). Final plasmid constructs were confirmed by DNAsequence analysis.

Construction of the Expression Plasmids

Antibody scFv encoding fragments were either digested directly frompBSK-B72.3, pBSK-CA19.9 or assembled from V_(H) and V_(L) domainsencoded by pASK19 plasmids respectively and inserted into NcoI, and NotIrestriction sites of previously digested plasmid pBAK1 to makepBAK1B72.3, pBAK1CA19.9 and pBAK14D5 respectively. The competent E. coliXL1 Blue cells were transformed using ligation mixtures and the cloneswere selected on the LB plates containing kanamycin. Positive cloneswere confirmed by DNA sequencing. To make RFP chimeras inV_(H)-RFP-V_(L) orientation, plasmids pBAK1B72.3, pBAK1CA19.9 andpBAK14D5 were digested with BamHI restriction enzyme and PCR product ofmRFP1 gene obtained using pMT-RFP plasmid template and oligonucleotideprimers RFPBamF and RFPBamR (Table 1), inserted to producepBAK1B72.3RFP, pBAK1CA19.9RFP and pBAK14D5RFP respectively. Colonieswere initially screened by colony PCR using primers T7F and RFPBamR(Table 1) and selected clones confirmed by plasmid DNA sequencing.

Antibody scFv encoding fragments of 4D5-8 were digested from plasmidpBAK14D5 and inserted into pA plasmid to make pA4D5. The competent E.coli XL1 Blue cells were transformed using ligation mixtures and theclones were selected on the LB plates containing carbenicillin. Positiveclones were confirmed by DNA sequencing. To make RFP, Citrine, andCerulean chimeras in V_(H)-FP-V_(L) orientation, this plasmid wasdigested with BamHI and the mRFP1 gene obtained using pMT-RFP plasmidtemplate and oligonucleotide primers RFPBamF and RFPBamR (Table 1), wasinserted to produce pA4D5RFP. The BamHI fragment encoding the mRFP wasreplaced by mCit and mCer to generate pA4D5mCit and pA4D5mCerrespectively. Colonies were initially screened by colony PCR usingprimers T7F and RFPBamR (Table 1) or CITBamR selected clones confirmedby plasmid DNA sequencing.

Antibody Expression in E. coli Periplasm.

To express scFv and REDantibody chimeras, E. coli BL21 (DE3) pRARE(Phage-resistant derivative of BL21 (DE3) (EMD Chemicals Inc.,Gibbstown, N.J.) cells were transformed with the appropriate plasmid andplated onto LB agar supplemented with kanamycin sulfate (30 μg/mL finalconcentration) and chloramphenicol (34 μg/mL final concentration). Thecells were allowed to grow at 37° C. for 18 hours and the following day,five fresh colonies were inoculated into 10 mL of LB media (withantibiotics) and grown at 37° C. (with shaking at 250 rpm) for 16 hours.Next day, 200 mL of pre-warmed LB media, prepared in 1 L conical flasks(with antibiotics) were inoculated with 10 mL of the overnight cultureand grown at 37° C. (with shaking at 250 rpm) until the optical densityat 600 nm had reached 0.5, then the cells were placed on ice for 30minutes and Isopropyl β-D-1-thiogalactopyranoside (IPTG) added (finalconcentration 0.3 mM) to the cultures and the cells were grown at 20° C.for an additional 20 hours with shaking at 250 rpm. Bacterial cells werepelleted by centrifugation for 20 minutes, 5,000 rpm at 4° C. (usingSorvall SuperT 21 bench top centrifuge, with SL-250T rotor) thesupernatant discarded and the pellets retained for periplasmic proteinextraction.

Antibody Expression in E. coli Cytoplasm.

To express scFv and REDantibody chimeras, in the cytoplasm of E. coliRosetta gami (DE3) (EMD Chemicals Inc., Gibbstown, N.J.) cells weretransformed with the appropriate plasmid and plated onto LB agarsupplemented with carbenicillin and chloramphenicol (100 μg/mL and 34μg/mL final concentration respectively). The cells were allowed to growat 37° C. for 18 hours and the following day, five fresh colonies wereinoculated into 10 mL of LB media (with antibiotics) and grown at 37° C.(with shaking at 250 rpm) for 16 hours. Next day, 200 mL of pre-warmedLB media, prepared in 1 L conical flasks (with antibiotics) wereinoculated with 10 mL of the overnight culture and grown at 37° C. (withshaking at 250 rpm) until the optical density at 600 nm had reached 0.5,then the cells were placed on ice for 30 minutes and Isopropylβ-D-1-thiogalactopyranoside (IPTG) added (final concentration 1 mM) tothe cultures and the cells were grown at 20° C. for an additional 20hours with shaking at 250 rpm. Fifty mL aliquots of bacterial cultureswere pelleted by centrifugation for 20 minutes, 5,000 rpm at 4° C.(using Sorvall SuperT 21 bench top centrifuge, with SL-250T rotor) thesupernatant discarded and the pellets retained for cytoplasmic proteinextraction.

Antibody Purification from E. coli Periplasm.

Bacterial cell pellets from 200 mL culture were resuspended in 10 mL ofperiplasmic buffer (30 mM Tris-base, pH 8.0, 20% sucrose and 1 mM EDTA)supplemented to a final concentration of 0.1 mM phenylmethylsulfonylfluoride (PMSF). The cells were incubated on ice for 10 minutes andcentrifuged at 9000 rpm for 10 minutes at 4° C. (using Sorvall SuperT 21bench top centrifuge, with SL-50T rotor). The supernatants werecollected and stored on ice, whilst cell pellets were resuspended in 7mL of 5 mM MgCl2 (4° C.). After incubation for five minutes on ice,bacterial cells were pelleted by centrifugation as described before, andthe supernatants combined to give the periplasmic fraction.

Antibody Purification from E. coli Cytoplasm.

Bacterial cell pellets from 50 mL culture were resuspended in 3 mL ofperiplasmic buffer (20 mM Tris-base, pH 8.0, 0.5 M NaCl, 10 mM imidazole0.1% Triton-X-100) supplemented to a final concentration of 0.1 mMphenylmethylsulfonyl fluoride (PMSF), lysozyme 0.2 mg/mL and sonicated6×30 second bursts. The cells were incubated on ice for 10 minutes andcentrifuged at 13000 rpm for 10 minutes at 4° C. (using Biofuge). Thesupernatants were collected to give the cytoplasmic fraction.

Ni-NTA Purification of Recombinant Proteins.

Recombinant proteins were purified using a 1 mL HisTrap HP column (GEHealthcare) fitted to an ÄKTAprime™ plus (GE Healthcare, Piscataway,N.J.) liquid chromatography system. First, the 1 mL column wasequilibrated with five column volumes of equilibration buffer (20 mMTris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole). Following this, 17 mLE. coli periplasmic fraction (diluted two times with 20 mM Tris-HCl, pH8.0, 500 mM NaCl buffer) or 3 mL of the cytoplasmic extract was loadedonto the column and the column was washed with five column volumes ofwash buffer (20 mM Tris-HCl, pH 8.0, 500 Mm NaCl, 20 mM imidazole). Theprotein was eluted with five column volumes of elution buffer (20 mMTris-HCl, pH 8.0, 500 mM NaCl, 500 mM imidazole) and collected in 1 mLaliquots. Protein elution was monitored at 280 nm. Eluted fractions 1-5were analysed by SDS-PAGE.

Desalting and Anion Exchange Chromatography

Recombinant antibody obtained by Ni-NTA purification were desalted using40 mL Sephadex G25 column (GE Healthcare, Piscataway, N.J.) fitted to anÄKTAprime™ plus (GE Healthcare, Piscataway, N.J.) liquid chromatographysystem in 20 mM Tris-HCl, pH 8.0 buffer and further purified using 1 mLHiTrap Q Sepharose FF column (GE Healthcare, Piscataway, N.J.) andeluted by increasing buffer salt concentration. First, a 1 mL column wasequilibrated with five column volumes of equilibration buffer (20 mMTris-HCl, pH 8.0). Following this, 5 mL of desalted antibody fractionwas loaded onto the column and protein was eluted by increasingconcentration of NaCl in 20 mM Tris-HCl, pH 8.0 buffer and collectedinto 1 mL fractions. Eluted antibodies were concentrated and the bufferexchanged to 20 mM Tris-HCl, pH 8.0 and 150 mM NaCl using Ultracel YM-10Amicon centrifugal devices from Millipore. Protein concentration wasdetermined using Bradford assay kit. (Bio-Rad Laboratories, Hercules,Calif.) (Bradford, 1976 Anal Biochem 72:248-54).

Size Exclusion Chromatography

SEC was performed on an ÄKTA Prime Plus using a HiLoad 16/60 Superdex200 size-exclusion column (GE Healthcare, Amersham, UK) equilibratedwith degassed phosphate buffered saline (PBS). The purified proteinswere separated by loading 1 mL of the sample at a concentration of100-200 μg/mL. The flow rate was 1 mL/min, and the absorbance of theeluted protein was monitored at 280 nm. The column was calibrated withthe following protein standards: β-amylase (200 kDa), bovine serumalbumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4kDa).

SDS-PAGE

The fractions eluted from the HisTrap column (1-5) were analysed bySDS-PAGE using 12% Tris-Glycine gels. Proteins were stained withCoomassie Blue 8250 as follows: after electrophoresis gel was submergedin plastic container into 50 mL of 0.025% Coomassie Blue in 10% aceticacid solution and heated in the microwave until boiling (approximately 1minute), cooled down for two minutes on bench and destained in 50 mL of10% acetic acid by repeating previous procedure of heating in microwaveand cooling down. Finally, the gel was kept in 10% acetic acid beforescanning.

Optical Properties Determination

The fluorescent measurements were carried out using SpectraMAX Gemini EMfluorescence microplate reader with Gemini EM software (MolecularDevices, Inc., Sunnyvale, Calif.) using Optiplex 96F microtitre plates(PerkinElmer, Inc., Waltham Mass.) with mRFP, mCIT, CER, and BFP 4D5-8antibodies, 50 μg in 0.1 mL/well in phosphate buffered saline (137 mMNaCl, 2.5 mM KCl, 10 mM Na₂HPO₄ and 1.8 mM KH₂PO₄) pH7.4. Excitation andemission spectra were determined at 1 nm intervals and the arbitraryfluorescence units (AFU) recorded. The concentration dependantfluorescence with excitation 584 nm and emission 607 nm was determinedfor ten-fold serial dilutions of REDantibody 4D5-8 from a stock solutionof 95 nmoles.

Surface Plasmon Resonance (SPR) Measurements

The SPR measurements were carried out on the BIAcore 3000 following theprocedure previously described to determine the binding of 4D5-8 scFvwith p185HER2-ECD (Worn and Pluckthun, 1998 FEBS Lett 427:357-61). Thep185HER2-ECD antigen (Sino Biological Inc; Beijing, China) 100 μg/mL in20 mM citrate buffer pH 4.0 was coupled to the CM-5 research gradesensor chip using an amine coupling kit (GE Healthcare, Piscataway,N.J.). The 4D5-8 REDantibody was applied at 50 μg/mL, 100 μg/mL, 175μg/mL, 250 μg/mL, and 500 μg/mL to the chip at a flow rate of 20 μL/minat 20° C. The surface was regenerated by injection of 45 μL of 0.1 Mglycine-HCL, pH 2.2, 0.5 M NaCl. Data were analyzed using the global fitin the BIAevaluation program version 4.1

Immunofluorescent Confocal Microscopy

Wild-type T. cruzi epimastigote four-days-old cultures were fixed with2% paraformaldehyde in phosphate buffer and allowed to adhere onto glassslides. Adhered cells were washed with PBS and non-specificantigen-binding sites were blocked with 2% BSA in PBS, pH 8.0, for 1hour at room temperature. Samples were incubated for 60 minutes at roomtemperature with recombinant REDantibodies. REDantibody 4D5 was used asa negative control. Slides were mounted in Npropylgalate (Sigma P130,Sigma-Aldrich, St. Louis, Mo.) to reduce photobleaching and observed ona Leica SP2 Confocal Laser Scanning Microscope, using 543-nm He—Nelaser. All images were analysed using IMARIS software version 6.3(Bitplane). Formaldehyde fixed Trypanosoma cruzi epimastigotes TC1strain were a gift from Professor Michael Miles and Michael Lewis.

Example 2 Monomeric Recombinant Fluorescent Antibody Platform for CellSorting and Molecular Imaging

Methods

Molecular Modelling.

X-ray structures of human Her2 with HERCEPTIN (Genentech, Inc., SouthSan Francisco, Calif.) antibody (PDB 1N8Z) and DsRed (PDB 1GyK) weredownloaded from RCSB Protein data bank. A mRFP1 model was generated byhomology-modeling using server Swiss-Model. MIFit program (RigakuAmericas Corp., The Woodlands, Tex.) was used to obtain a molecular 3Dmodel of human Her2 with HERCEPTIN (Genentech, Inc., South SanFrancisco, Calif.) Fv domains and mRFP1. The 3D model of the proposedrefined structure 4D5-8mRFP1 was generated using PyMOL software.

Construction of the Expression p4D5-8mRFP1 Vector.

Genetic engineering manipulations, plasmid preparation, cell culture,protein expression and cell lysis followed the standard protocols.

Primers are shown in Table 3.

TABLE 3 Primer Sequence SEQ ID NO: 4D5BAMF 5′-GTCCCTTCTCGCTTCTCTGGGTSEQ ID NO: 9 CCAGATCTGGGACGGATTTCAC-3′ 4D5BAMR 5′-GTGAAATCCGTCCCAGATCTGGSEQ ID NO: 10 ACCCAGAGAAGCGAGAAGGGAC-3′ PAKVLBAM5′-CAGCGGCGGAGGCGGATCCGAT SEQ ID NO: 11 ATCCAGATGACCCAGTC-3′ PAKVLNOT5′-TCGAGTGCGGCCGCATCCGCGC SEQ ID NO: 12 GTTTGATCTCCACCTTGGTAC-3′PAKVHBAM 5′-TCGGATCCGCCTCCGCCCGAGG SEQ ID NO: 13 AGACGGTGACCAGGGTTC-3′PAKVHNCO 5′-TAGGCCATGGCCGAGGTTCAGC SEQ ID NO: 14 TGGTGGAG-3′ RFPBamF5′-CAGTGGATCCGAGGACGTCATC SEQ ID NO: 15 AAGGAGTTC-3′ RFPBamR5′-CAGTGGATCCGCCTCCGCCTGT SEQ ID NO: 16 GCGGCCCTCGGCGCGCTCGTAC-3′CITBamR CAGTGGATCCGCCGCCGCCGGTAAT SEQ ID NO: 17 GCCCGCCGCGGTCACAll primers were purchased from Invitrogen Corp. (Carlsbad, Calif.).Plasmids pAK19 was provided by Dan Yansura (Genentech, Inc., South SanFrancisco, Calif.), pMT-RFP and pMT-BFP were provided by Ray St. Ledger(University of Maryland, College Park, Md.). Restriction nuclease NcoI,Nod and BamHI, T4 DNA ligase, CIP were purchased from New EnglandBiolabs, Inc. (Ipswich, Mass.). The mRFP1 was amplified by PCR frompMT-RFP plasmid using primers RFPBamF and RFPBamR. The V_(H) and V_(L)domains of the humanized humAb 4D5-8 antibody were amplified by PCR fromthe plasmid pAK19 using PAKVHNCO/PAKVHBAM and PAKVLBAM/PAKVLNOTrespectively. To obtain the scFv of the humanized 4D5-8 antibody andsimultaneously introduce BamHI restriction site between V_(H) and V_(L)chains, PCR amplified V_(H) and V_(L) chains were joined bysplice-overlapping PCR using PAKVHNCO and PAKVLNOT. Internal BamHI sitepresent in the original V_(L) chain of humAb4D5-8 was eliminated bytwo-step mutagenesis PCR using primers 4D5BAMF and 4D5BAMR. Finalproduct of scFv 4D5-8 was digested with NcoI and NotI restrictionenzymes and inserted into the same sites of pBAK1 plasmid in frame withpelB leader sequence and an octa His-tag to make p4D5-8Bam plasmid. Thisplasmid was subsequently digested with BamHI and mRFP1 DNA inserted toobtain p4D5-8mRFP1 plasmid. All constructs were verified by DNAsequencing.Protein Expression.

Transformants from strain BL21 (DE3) pRARE (Phage-resistant derivativeof BL21(DE3), with pRARE plasmid encoding rare codon tRNAs,chloramphenicol-resistant) were inoculated into 200 mL Luria-Bertani(LB) medium plus kanamycin (30 μg/mL) and chloramphenicol (34 μg/mL),and grown overnight at 37° C. This culture was used to inoculate a 12 Lfermentor (1:60 dilution) containing LB medium plus kanamycin (30 μg/mL)and chloramphenicol (34 μg/mL), and grown for approximately 5 hours at37° C. until optical density (OD_(600nm)) of the culture reached 0.6.Then Isopropyl-β-D-thio-galactoside (IPTG) at the final concentration of0.3 mM was added to the culture to induce protein expression.Fermentations were performed at 20° C. for 20 hours at 350 rpm, four gasvolume flow per unit of liquid volume per minute (vvm) aeration at pH7.2.

Protein Purification and Analysis.

Protein was prepared from the whole cell lysates, purified on 1 mLHisTrap HP Ni Sepharose™ column using ÄKTAprime™ plus purificationsystem (GE Healthcare, Piscataway, N.J.) and analyzed by two 12%SDS-PAGE Tris-Glycine gels. One gel was stained with Coomassie and theother was used for Western immunoblots. The cells were harvested, washedwith PBS, centrifuged, and the pellet was resuspended in lysis buffer(0.01 M Tris HCl pH 8.0, containing 0.5 M NaCl, 10 mM imidazole and 0.1%(vol/vol) Triton X100) and sonicated on ice. The lysate was thencentrifuged at 22,000×g for 30 minutes at 4° C. The supernatant was usedfor the purification of His-tagged proteins using immobilized metal ionaffinity chromatography (IMAC) and proteins were eluted with 500 mMimidazole. The metal affinity-enriched proteins were loaded ontoSephadex G200 gel filtration column (GE Healthcare, Piscataway, N.J.)run using 0.02 M Tris HCl pH 7.5, containing 0.15 M NaCl, with a flowrate of 1 mL/min, collecting 1 mL fractions. Fractions 32-37 were pooledand concentrated using Microcon concentrators (Millipore, Billerica,Mass.) with a molecular mass cutoff of 10 kDa, and verified by SDS-PAGEand Western blot analysis. SDS/PAGE analyses were performed according tothe standard protocols using 12% polyacrylamide gels. Immunoblots onnitrocellulose membrane (Millipore, Billerica, Mass.) were carried outaccording to the manufacturer's instructions using monoclonal anti-polyhistidine-alkaline phosphatase antibody clone His-1 (Sigma A5588,Sigma-Aldrich, St. Louis, Mo.) were visualized using an5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT)(Sigma B1911, Sigma-Aldrich, St. Louis, Mo.).

Flow Cytometry and Fluorescence Microscopy Analysis.

The SKBr-3 and MDA-MB-231 cell lines were maintained in RPMI medium1640, supplemented with 10% FCS and 2 mM L-glutamine in culture flasks.For flow cytometry analysis, the adherent SKBr-3 and MDA-MB-231 cellswere carefully detached with trypsin in PBS (pH 8.0) containing 5 mMEDTA. After a brief wash with cold PBS the cells were incubated with0.2% paraformaldehyde in PBS for five minutes and again washed with coldPBS, counted, incubated with 4D5-8mRFP1 30 minutes on ice, washed twicewith cold buffer Dulbecco's PBS containing 1% FCS, 2 mM EDTA (pH 7.2)(MACS), and analyzed by CyAn™ ADP flow cytometer (Dako North America,Inc., Carpinteria, Calif.). Results were analyzed using Summit softwarefrom Beckman Coulter. For immunofluorescent analysis, cells were platedon glass cover slips at density of 4×10³ cells per well and culturedovernight at 37° C. in a 5% CO2 atmosphere. Cells were fixed in 0.2%paraformaldehyde in PBS for five minutes, washed with PBS 3x,permeabilized with 0.1% Triton-X 100 in PBS for five minutes, washed asbefore, incubated with 4D5-8mRFP1 (10 μg/mL) and GelGreen™ nuclear stain(Biotium, Inc., Hayward, Calif.) for 30 minutes, washed as before andanalyzed using fluorescence microscope Axioscop 50 (Carl ZeissMicroImaging, LLC; Thornwood, N.Y.). Images were captured by using a CCDcamera (PowerShot digital camera, Canon U.S.A., Inc., Lake Success,N.Y.) and AxioVision software (Carl Zeiss MicroImaging, LLC; Thornwood,N.Y.).

Molecular Design and Visualisation

Structure of B72.3 and 4D5 antibodies were downloaded from PDB database(PBD: 1BBJ and 1FVC respectively). RFP structure was predicted usingSwiss-Model Workspace server. Further modeling was performed usingMIFit+ software version 2009.09-1 (Rigaku Americas Corp., The Woodlands,Tex.) and protein models were viewed using PyMOL software version 1.1(DeLano Scientific LLC, Palo Alto, Calif.).

Plasmids, Primers and Synthetic DNA

Plasmid pBAK1, previously constructed in our laboratory is based onpET-26b vector (EMD Chemicals Inc., Gibbstown, N.J.). All primers werepurchased from Invitrogen Corp. (Carlsbad, Calif.). Synthetic DNAsequences of B72.3 and CA19.9 antibody variable domains in V_(H)-V_(L)orientation were codon optimized for E. coli expression and purchasedfrom Epoch Biolabs, Inc. (Missouri City, Tex.) as plasmids pBSK-B72.3and pBSK-CA19.9, respectively, with NcoI, BamHI and NotI restrictionsites to facilitate construction of the expression vectors. PlasmidpAK19 encoding the 4D5-8 V_(H) and V_(L) was provided by Dan Yansura(Genentech, Inc., South San Francisco, Calif.). Plasmid pMT-RFP withmRFP1 gene was a gift from Professor Ray St. Ledger.

Bacterial Strains, Growth Media and Recombinant DNA Technique

XL1-Blue Escherichia coli strain (Stratagene, Agilent Technologies,Inc., Santa Clara, Calif.) was used for plasmid construction steps. Toexpress recombinant antibodies BL21 (DE3) strain of E. coli (EMDChemicals Inc., Gibbstown, N.J.) were used. E. coli cells were grown inLysogeny Broth (LB) (Bertani, 2004 J Bacteriol 186:595-600) or LB agarplates. Kanamycin sulfate and carbenicillin were used at 30 μg/mL and100 μg/mL final concentrations respectively. Plasmid DNA was isolatedusing QIAprep Spin Miniprep Kit (Qiagen Inc.—USA, Valencia, Calif.) andDNA from gel was purified using QIAquick Gel Extraction Kit (QiagenInc.—USA, Valencia, Calif.). E. coli cells were transformed usingstandard heat shock methods. Restriction and modification enzymes werepurchased from New England Biolabs, Inc. (Ipswich, Mass.). Final plasmidconstructs were confirmed by DNA sequence analysis.

Construction of the Expression Plasmid

Antibody scFv encoding fragments were either digested directly frompBSK-B72.3, pBSK-CA19.9 or assembled from V_(H) and V_(L) domainsencoded by pASK19 plasmids respectively and inserted into NcoI, and NotIrestriction sites of previously digested plasmid pBAK1 to makepBAK1B72.3, pBAK1CA19.9 and pBAK14D5 respectively. The competent E. coliXL1 Blue cells were transformed using ligation mixtures and the cloneswere selected on the LB plates containing kanamycin. Positive cloneswere confirmed by DNA sequencing. To make RFP chimeras inV_(H)-RFP-V_(L) orientation, plasmids pBAK1B72.3, pBAK1CA19.9 andpBAK14D5 were digested with BamHI restriction enzyme and PCR product ofmRFP1 gene obtained using pMT-RFP plasmid template and oligonucleotideprimers RFPBamF and RFPBamR (Table 1), inserted to producepBAK1B72.3RFP, pBAK1CA19.9RFP and pBAK14D5RFP respectively. Colonieswere initially screened by colony PCR using primers T7F and RFPBamR(Table 1) and selected clones confirmed by plasmid DNA sequencing.

Antibody Expression in E. coli.

To express scFv and REDantibody chimeras, E. coli BL21 (DE3) (EMDChemicals Inc., Gibbstown, N.J.) cells were transformed with theappropriate plasmid and plated onto LB agar supplemented with kanamycinsulfate (30 μg/mL final concentration). The cells were allowed to growat 37° C. for 18 hours and the following day, five fresh colonies wereinoculated into 10 mL of LB media (with antibiotics) and grown at 37° C.(with shaking at 250 rpm) for 16 hours. Next day, 200 mL of pre-warmedLB media, prepared in 1 L conical flasks (with antibiotics) wereinoculated with 10 mL of the overnight culture and grown at 37° C. (withshaking at 250 rpm) until the optical density at 600 nm (OD₆₀₀) hadreached 0.5, then the cells were placed on ice for 30 minutes andIsopropyl β-D-1-thiogalactopyranoside (IPTG) added (final concentration0.3 mM) to the cultures and the cells were grown at 20° C. for anadditional 20 hours with shaking at 250 rpm. Bacterial cells werepelleted by centrifugation for 20 minutes, 5,000 rpm at 4° C. (usingSorvall SuperT 21 bench top centrifuge, with SL-250T rotor), thesupernatant discarded, and the pellets retained for periplasmic proteinextraction.

Antibody Purification from E. coli Periplasm.

Bacterial cell pellets from 200 mL culture were resuspended in 10 mL ofperiplasmic buffer (30 mM Tris-base, pH 8.0, 20% sucrose and 1 mM EDTA)supplemented to a final concentration of 0.1 mM phenylmethylsulfonylfluoride (PMSF). The cells were incubated on ice for 10 minutes andcentrifuged at 9000 rpm for 10 minutes at 4° C. (using Sorvall SuperT 21bench top centrifuge, with SL-50T rotor). The supernatants werecollected and stored on ice; cell pellets were resuspended in 7 mL of 5mM MgCl₂ (4° C.). After incubation for five minutes on ice, bacterialcells were pelleted by centrifugation as described above, and thesupernatants combined to give the periplasmic fraction.

Ni-NTA Purification of Recombinant Proteins.

Recombinant proteins were purified using a 1 mL HisTrap HP column (GEHealthcare, Piscataway, N.J.) fitted to an ÄKTAprime™ plus (GEHealthcare, Piscataway, N.J.) liquid chromatography system. First, the 1mL column was equilibrated with five column volumes of equilibrationbuffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole). Followingthis, 17 mL E. coli periplasmic fraction (diluted two times with 20 mMTris-HCl, pH 8.0, 500 mM NaCl buffer) was loaded onto the column and thecolumn was washed with five column volumes of wash buffer (20 mMTris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole). The protein was elutedwith five column volumes of elution buffer (20 mM Tris-HCl, pH 8.0, 500mM NaCl, 500 mM imidazole) and collected in 1 mL aliquots. Proteinelution was monitored at 280 nm. Eluted fractions 1-5 were analyzed bySDS-PAGE.

Desalting and Anion Exchange Chromatography

Recombinant antibody obtained by Ni-NTA purification were desalted using40 mL Sephadex G25 column (GE Healthcare, Piscataway, N.J.) fitted to anÄKTAprime™ plus (GE Healthcare, Piscataway, N.J.) liquid chromatographysystem in 20 mM Tris-HCl, pH 8.0 buffer and further purified using 1 mLHiTrap Q Sepharose FF column (GE Healthcare, Piscataway, N.J.) andeluted by increasing buffer salt concentration. First, a 1 mL column wasequilibrated with five column volumes of equilibration buffer (20 mMTris-HCl, pH 8.0). Following this, 5 mL of desalted antibody fractionwas loaded onto the column and protein was eluted by increasingconcentration of NaCl in 20 mM Tris-HCl, pH 8.0 buffer and collectedinto 1 mL fractions. Eluted antibodies were concentrated and the bufferexchanged to 20 mM Tris-HCl, pH 8.0 and 150 mM NaCl using Ultracel YM-10Amicon centrifugal devices (Millipore, Billerica, Mass.). Proteinconcentration was determined using Bradford assay kit (Bio-RadLaboratories, Hercules, Calif.) (Bradford, 1976 Anal Biochem 72:248-54).

Size Exclusion Chromatography

Size exclusion chromatography was performed on an ÄKTA Prime Plus usinga HiLoad 16/60 Superdex 200 size-exclusion column (GE Healthcare,Piscataway, N.J.) equilibrated with degassed phosphate buffered saline(PBS). The purified proteins were separated by loading 1 mL of thesample at a concentration of 100-200 μg/mL. The flow rate was 1 mL/min,and the absorbance of the eluted protein was monitored at 280 nm. Thecolumn was calibrated with the following protein standards: β-amylase(200 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa)and cytochrome c (12.4 kDa).

SDS-PAGE

The fractions eluted from the HisTrap column (1-5) were analysed bySDS-PAGE using 12% Tris-Glycine gels. Proteins were stained withCoomassie Blue 8250 as follows: after electrophoresis gel was submergedin plastic container into 50 mL of 0.025% Coomassie Blue in 10% aceticacid solution and heated in the microwave until boiling (approximately 1min), cooled down for two minutes on the bench and destained in 50 mL of10% acetic acid by repeating previous procedure of heating in microwaveand cooling down. Finally, the gel was kept in 10% acetic acid beforescanning.

Example 3 Delivery of Recombinant P. agglomerans to the Cibarium of H.vitripennis

For this experiment, one can use adult H. vitripennis collected fromcitrus orchards at the Agricultural Operations at UC Riverside.Experimental group plants can be coated with recombinant P. agglomeranscontaining a plasmid encoding a fluorescent antibody such as, forexample, the REDantibody described herein. Control group plants can becoated with bacteria-free growth medium.

To verify that sharpshooters from the Agricultural Operations are freeof recombinant P. agglomerans in their natural state, one can analyze P.agglomerans cibarial tissues using PCR and fluorescence microscopy. Foreach trial, one can use 10 potted Chardonnay grape vines of 3 years' agethat exhibit new growth. The shoots of each plant can be hand paintedwith liquid cultures of recombinant P. agglomerans at a concentration of10⁸ CFU/mL to achieve coverage of 100 percent surface area. Applicationof recombinant P. agglomerans cultures can occur with 1.0 mL/cm² ofplant surface area (shoots only). In the set-up of this trial, one cantrim the 10 plants to allow approximately 200 cm² of shoot surface area.Thus, one delivers approximately 200 mL or close to 2×10¹⁰ CFU ofrecombinant P. agglomerans to each plant.

Sharpshooters (n=100) can be introduced into the cage and allowed tofeed for 5 days. At this early stage, one observes little mortality andone can remove all sharpshooters from the cage and place them in a newcage containing five untreated plants. At transfer, 20 sharpshooters canbe sacrificed and cibarial tissues can be dissected out. Analysis ofcolonization by recombinant P. agglomerans can be conducted using threemethods: (1) confocal microscopy to detect Red Fluorescent Protein at608 nm (2) qtPCR with primers specific for P. agglomerans, and (3)standard light microscopy for visualization of red bacteria. Theremaining 80 insects can be serially analyzed over the next 20 days forretention of recombinant P. agglomerans in the cibarium.

Recombinant P. agglomerans will colonize the cibarium of H. vitripennis.In the initial 20 sharpshooters that are removed after five days,uniform colonization of the anterior mouthparts of H. vitripennis willbe observed. The outcome of cibarial colonization can be measured byfluorescence/confocal microscopy, qtPCR, and/or light microscopy forvisualization of red pigment. Two specific outcomes can be measured: (1)percent of sharpshooters that carry recombinant P. agglomerans in thecibarium, and (2) relative microbial CFU in the sharpshooters that arepositive for recombinant P. agglomerans. Other environmental bacteriamay colonize the mouthparts of H. vitripennis and microbial competitioncan occur. One can measure total bacterial load by performing qtPCR withuniversal 16S RNA primers and measure the fraction of total bacterialburden that is attributed to recombinant P. agglomerans. Theexperimental parameters of this study—i.e., 100 percent coverage ofshoots with 10⁸ CFU of recombinant P. agglomerans per mL of appliedculture—are established to optimize conditions for colonization.Statistical significance of recombinant P. agglomerans colonization inexperimental group sharpshooters can be determined by comparing qtPCRvalues with control insects by chi squared analysis.

The second part of this study, persistence of recombinant P. agglomeransafter initial exposure can reveal colonization of the cibarium inexperimental group sharpshooters. There may be some decrease in CFU overthe 20-day period, but one can calculate rates of decay via qtPCR. Thedecay rate can adjust for plasmid loss of the engineered recombinant P.agglomerans.

Transmission Blocking Effect of REDantibody in GWSS

One can assess the effect of REDantibody, expressed by recombinant P.agglomerans in paratransgenic H. vitripennis, in: (1) rendering thearthropod refractory to challenge infection by X. fastidiosa, and (2)preventing transmission of the pathogen to healthy grape vines. Threegroups of fourth-instar GWSS can be used: (1) an experimental group canbe colonized with recombinant P. agglomerans as above (n=100), (2) acontrol group of GWSS can be exposed to P. agglomerans transformed toexpress a marker antibody (rDB3) that has no activity against Xylella(N=100), (3) a second control can consist of GWSS exposed tountransformed P. agglomerans.

For these studies, we can use freshly molted adults to increaselikelihood of Xylella transmission. Sharpshooters can be exposed to P.agglomerans-coated Chardonnay grape plants for five days. Ten insectsfrom each group can be sacrificed at day 5 to confirm cibarialcolonization by P. agglomerans and expression of antibody (ELISA,Western blot). Using the protocol of Almeida and Purcell (Almeida, R. P.and Purcell, A. H., Transmission of Xylella fastidiosa to grapevines byHomalodisca coagulata (Hemiptera: Cicadellidae). J Econ Entomol, 2003.96:264-71), remaining insects can be challenged with X. fastidiosa.Briefly, GWSS that have been exposed to P. agglomerans can betransferred to closed containers containing Xylella-infected Chardonnaygrape (confirmed by culture and PCR of plant issues). Seedlings withsucculent growth can be used throughout to maximize feeding andtransmission. The Acquisition Access Period (AAP) can be two days. Thiscan permit acquisition of Xylella by the nymphs and prevent excessiveloss of the recombinant P. agglomerans. Following the two-day AAP, 10insects from each group can be sacrificed and cibarial tissues can beexamined by both culture and PCR for X. fastidiosa. Since presence ofXylella alone in GWSS is not correlated to subsequent transmission, wecan assess infectivity of the three groups of insects by transferringthem to fresh, uninfected grape plants with succulent growth for anInoculation Access Period (IAP) of 7-21 days, until molting occurs. Thiscan allow ample time for transmission to occur and, possibly, cycles oftransmission to occur between plants. Plants from the previous step canbe stored separately and monitored for a minimum of eight weeks toassess for symptoms of Pierce's Disease. All plants can be examined byculture and PCR to verify X. fastidiosa infection.

Insects in the two control groups will be infected with Xylella at ratesof nearly 100 percent. Likewise, between 80-90% transmission of thepathogen and disease in target grape vines are seen in the controlgroups. Transmission of Xylella and infection of grape plants after theIAP, however, will be reduced by approximately 30%, preferably byapproximately 50%, in the experimental group of sharpshooters.

Example 4 Survival and Dispersal of pSCR-189b-Transformed (Recombinant)P. agglomerans in the Rhizosphere of Chardonnay Grape Vines

One can measure soil CFU of recombinant P. agglomerans before and aftereach 25-day trial for each of the experimental pots containingrecombinant P. agglomerans-treated grape plants. Five separate soilsamples (1.0 cm³) can be taken from the base of the grape vine at days1, 12, and 25. Soil analysis for recombinant P. agglomerans can beconducted using one or more of three techniques: (1) culture onselection agar with PCR identification of colonies (gold standard), (2)fluorescence microscopy to detect RFP, and/or (3) direct visualizationof colonies on agar for red color. Analytic techniques can evaluate notonly the persistence and dispersal of recombinant P. agglomerans intothe rhizosphere but also the accuracy of direct color visualization ofbacterial colonies to identify recombinant bacteria.

No recombinant P. agglomerans are present in the rhizosphere on day 1.Probing of shoots by GWSS, physical detachment of bacteria from shootsand movement of bacteria through watering activities may to contributeto rhizosphere invasion by recombinant P. agglomerans. Therefore, CFUcounts of recombinant P. agglomerans may increase by day 12. Values onday 25 can reflect a balance of bacterial replication and gene decay.Comparison of values with control pots using chi-squared analysis canyield measures of statistical significance.

Definitive detection of recombinant P. agglomerans may be performedusing colony PCR derived from selection agar, but may be a cumbersomeand expensive method for field use to detect paratransgenics. CFU countsof red bacteria can initially be compared statistically to valuesobtained by colony PCR to determine the accuracy of direct visualizationof color. Similar comparison can be made to fluorescent readouts thatdetect mRFP. Certain factors may decrease accuracy of visualizationmethods. First, other soil bacteria may appear red on agar (e.g.,Serratia spp., Gordonia spp.). Second, subjective variability canconfound the readings. Third, intensity of red color can wane over time.All readings that involve direct visualization of colonies on agar canbe repeated by a blinded observer to minimize bias. Concordance betweendirect visual methods and PCR data can be used to determine accuracy ofthis method.

Extent of Horizontal Gene Transfer Between Recombinant Pa andRhizosphere Bacteria

Populations of recombinant P. agglomerans can be exposed to a variety ofmicrobes commonly found in soil consortia to determine the extent ofhorizontal gene transfer (HGT). HGT is a dynamic process and accountsfor significant portions of the genomes of bacteria such as E. coli.Conjugative transfer of plasmids, heterologous recombination ofchromosomal material, retrotransposons, and phage-mediated gene flow aresome of the mechanisms by which microbes exchange genetic material.

HGT may occur in this milieu and one can assess the ability to monitorHGT events using the REDantibody platform. Though HGT has contributed toplasticity of microbial genomes, individual events are relativelyinfrequent and result from bacterial encounters in the 10⁷⁻⁸ range. Onecan score events in these experiments by counting CFUs of recipientbacteria that acquire the transgene using one or more of: (1) selectionand PCR-based assay of REDantibody gene, and/or (2) direct visualizationfor red color. Control exchanges can involve direct co-incubation ofwild-type P. agglomerans with consortium bacteria. Statisticalsignificance of the difference in events—e.g., HGT from control versusrecombinant P. agglomerans, and quantification of bacteria usingPCR-based analysis versus direct color visualization—can be calculatedusing chi-squared analysis.

Recombinant P. agglomerans expressing bright red color can beco-incubated with representative bacteria of the rhizosphere of commonagricultural plants. Though rhizosphere analysis reveals highly variedstructure and variability over time, one can choose five representativeorganisms that are commonly found in soil environments: Pseudomonasfluorescens, Arthrobacter globiformis, Escherichia coli, Enterobacteragglomerans, and Acinetobacter sp. For these studies, one may elect toinclude only Gram-negative bacteria with similar lipid bilayer membranesas P. agglomerans. Though Gram-positive bacteria with peptidoglycanmembranes and eukaryotic organisms such as yeast also may be present inthe rhizosphere, one can exclude them from this analysis since directvisualization of red color is an endpoint.

One can first verify that the target organisms lack red color.Recombinant P. agglomerans can serve as the donor organism at a totalCFU count of 10¹² bacteria/reaction in liquid culture. Recipientbacteria can be co-incubated at 22° C. at equal CFU count for timepoints of 24 hours, 48 hours, 96 hours, or 168 hours. Cultures forampicillin-resistant recipient bacteria can be done on selective agarand number of recombinant events can be scored. Identity of recombinantbacteria can be determined as above using 16S RNA-specific primers forPCR and compared to direct visualization for color.

Both invasion of the rhizosphere by recombinant P. agglomerans and HGTbetween recombinant P. agglomerans and consortium members may beobserved. Indeed, the large numbers of recombinant P. agglomerans usedin these trials, the protracted nature of these experiments, and thefavorable conditions for HGT are designed to promote gene spread in therhizosphere. One endpoint is evaluation of accuracy of direct colorvisualization to determine the extent of rhizosphere invasion and HGT inthese confined settings.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. As used herein, theterm “include” and its grammatical variants are intended to benon-limiting, such that recitation of items in a list is not to theexclusion of other like items that can be substituted or other itemsthat can be added to the listed items.

Upon studying the disclosure, it will be apparent to those skilled inthe art that various modifications and variations can be made in thedevices and methods of various embodiments of the invention. Otherembodiments will be apparent to those skilled in the art fromconsideration of the specification and practice of the embodimentsdisclosed herein. It is intended that the specification and examples beconsidered as examples only. The various embodiments are not necessarilymutually exclusive, as some embodiments can be combined with one or moreother embodiments to form new embodiments.

What is claimed is:
 1. A method of detecting an analyte comprising:providing a sample that comprises the analyte; contacting the samplewith a polypeptide that comprises: a fluorescent domain comprising: amonomeric fluorescent polypeptide comprising a C-terminus; and anN-terminus; a first antibody domain covalently linked to the C-terminusof the fluorescent domain, wherein the first antibody domain comprises avariable light chain (V_(L)) or a variable heavy chain (V_(H)); and asecond antibody domain covalently linked to the N-terminus of thefluorescent domain, wherein the second antibody domain comprises avariable light chain (V_(L)) or a variable heavy chain (V_(H)); whereinthe N-terminus of first antibody domain and the C-terminus of the secondantibody domain are separated by a distance of no less than 30 Å and nomore than 40 Å, wherein at least one of the first antibody domain andthe second antibody domain specifically binds to the analyte; removingunbound polypeptides; and detecting a fluorescent signal produced by thepolypeptide specifically bound to the analyte, thereby detectingpresence of the analyte in the sample.
 2. The method of claim 1 furthercomprising immobilizing at least a portion of the sample on a substrate.3. The method of claim 1 further comprising: contacting at least aportion of the sample with a second polypeptide that comprises: a secondfluorescent domain comprising: a second monomeric fluorescentpolypeptide comprising a C-terminus; and an N-terminus; a third antibodydomain covalently linked to the C-terminus of the second fluorescentdomain, wherein the third antibody domain comprises a variable lightchain (V_(L)) or a variable heavy chain (V_(H)); and a fourth antibodydomain covalently linked to the N-terminus of the second fluorescentdomain, wherein the fourth antibody domain comprises a variable lightchain (V_(L)) or a variable heavy chain (V_(H)); wherein the N-terminusof third antibody domain and the C-terminus of the fourth antibodydomain are separated by a distance of no less than 30 Å and no more than40 Å, wherein at least one of the third antibody domain and the fourthantibody domain specifically binds to a second analyte in the sample;removing unbound second polypeptide; and detecting fluorescent signalproduced by the second polypeptide specifically bound to the secondanalyte, thereby detecting presence of the second analyte in the sample.4. The method of claim 1 further comprising quantifying the fluorescentsignal.